Modulation of Host Cell Apoptosis by Trypanosoma cruzi: Repercussions in the Development of Chronic Chagasic Cardiomyopathy

Abstract

Trypanosoma cruzi is an intracellular parasite, which causes Chagas disease, affecting millions of people throughout the world. T. cruzi can invade several cell types, among which macrophages and cardiomyocytes stand out. Chagas disease goes through two stages: acute and chronic. If it becomes chronic, its most severe form is the chagasic chronic cardiomyopathy, which accounts for most of the fatalities due to this disease. For parasites to persist for long enough in cells, they should evade several host immune responses, one of these being apoptosis. Apoptosis is a type of programmed cell death described as a well-ordered and silent collection of steps that inevitably lead cells to a noninflammatory death. Cells respond to infection by initiating their own death to combat the infection. As a result, several intracellular microorganisms have developed different strategies to overcome host cell apoptosis and persist inside cells. It has been shown that T. cruzi has the ability to inhibit host cells apoptosis and can also induce apoptosis of cells that combat the parasite such as cytotoxic T cells. The aim of this chapter is to present up-to-date information about the molecules and mechanisms engaged by T. cruzi to achieve this goal and how the modulation of apoptosis by T. cruzi reflects in the development of chronic chagasic cardiomyopathy.

1. Introduction

The long-neglected disease called American trypanosomiasis or Chagas disease (CD) was first described in 1909 by the Brazilian doctor Carlos Chagas and it currently affects millions of people throughout the world. Its causative agent is the protozoan parasite Trypanosoma cruzi (Kinetoplastidae: Trypanosomatidae) found in more than 150 species of domestic and wild mammals that act as reservoirs [1, 2]. There are several transmission routes to humans such as the inoculation of parasites present in the feces of a hematophagous insect vector of the Triatominae subfamily or consumption of contaminated food with these feces, blood transfusions, organ transplants, and congenital [3]. The infection has a self-limiting acute phase with evident or absent parasitemia and may go unnoticed in many infected individuals. Some patients succumb during the acute phase of the disease, while others develop an adaptive immune response that generally controls the infection. If the parasite cannot be completely eradicated, the infection can last a lifetime and, if left untreated, presents potentially life-threatening complications such as chronic chagasic cardiomyopathy (CCC), which can present itself in its acute form or after a latency period that can extend for decades [4]. The persistence of the parasite in the host for such long periods indicates that it must surpass the host’s immune response mechanisms, of which apoptosis is among the most important. Interestingly, T. cruzi is capable of inducing or preventing apoptosis of host cells as needed [5]. In this work, we describe the basic aspects of Chagas disease and analyze the role of apoptosis in the infection by this protozoan parasite.

2. Epidemiology

CD is endemic in 21 countries in Latin America, from Mexico to the south of Argentina and Chile. Nevertheless, due to migrations and climate change, the disease has spread alarmingly to other parts of the world [6]. The World Health Organization (WHO) classifies this parasitic disease as a VBD (Vector-Borne Disease) that is among the unattended tropical diseases associated to extreme poverty in rural areas where the vectors are distributed, which favors their transmission route [7]. VBDs can be caused by bacteria, virus, and parasites, are usually transmitted by bloodsucker arthropods, represent 17% of all infectious diseases, and are prevalent in tropical and subtropical regions [8]. During the past three decades, the epidemiological overview of this disease has experienced important changes due to the implementation of vector control measures such as the use of pesticides and housing improvements [9]. In this respect, there has been a clear descent in the number of people infected by T. cruzi, going from 30 million in 1990 to 6–7 million currently infected. The incidence has also decreased from 700,000 estimated cases in 1990 to 30,000 cases per year in 2018. Furthermore, the mortality has gone down from 45,000 to 12,000 deaths in the same years [10, 11]. Despite all these efforts, CD continues to be an important health problem. Migration of asymptomatic people in chronic stage, who are unaware of their infection, has led to the spread of the disease to urban areas and nonendemic regions, increasing the frequency of cases in countries such as the United States, Japan, Australia, Spain, Italy, the United Kingdom, and other European countries where it is considered an emerging disease [6, 12]. In Latin America, Argentina (1,535,235), Brazil (1, 156, 821), and Mexico (876, 458) are the countries with the highest number of cases, followed by Bolivia with 607, 186 cases, making it the country with the highest prevalence, with an estimate between 6.8 and 18% of the population seropositive for T. cruzi [13]. Recent estimations suggest that there could be 300,000 infected people with T. cruzi in the United States [14]. A study performed in Los Angeles reported a seroprevalence of 5.2% among Latin American migrants who also presented cardiac abnormalities [15]. Outside of America, Spain is the most affected country. It is estimated that only in this country there could be more than 50,000 infected individuals, most of them South American immigrants [16].

3. Trypanosoma cruzi life cycle

T. cruzi goes through several developmental stages. Within the vector, it is possible to find epimastigotes and metacyclic trypomastigotes, while blood trypomastigotes and amastigotes are found in vertebrates [17]. The cycle starts when a triatomine acquires blood trypomastigotes by sucking blood from an infected mammal [2]. Inside the vector, in the medial posterior gut, trypomastigotes differentiate to epimastigotes and duplicate. Afterward, epimastigotes migrate to the rectum, where they differentiate to metacyclic trypomastigotes. During the bloodmeal, vectors defecate and expel metacyclic trypomastigotes in their droppings that infect the mammal through the site of the bite, fissures in the skin, or mucous membranes [3, 18]. Parasites invade macrophages and connective tissue cells close to the site of entry due to the action of surface glycoproteins involved in the anchoring/penetration process such as gp82, gp83, gp85, and Tc-85, as well as glycoinositolphospholipids (GIPLs) [19, 20, 21, 22, 23]. Once inside cells, parasites have the ability to escape from the phagolysosome because of the action of enzymes such as a trans-sialidase (TS) and a low pH-dependent pore-forming protein, which allow them to reach the cytosol, where metacyclic trypomastigotes differentiate to amastigotes, duplicate, and finally transform to blood trypomastigotes that lyse the cell [24, 25]. They have the ability to surpass host effector mechanisms due to the expression of surface molecules such as GP160 and calreticulin that allow them to evade the action of complement, and GPI-mucins, which act as an antigenic protective coat. This antigenic coat presents variations that partially explain the differences in virulence and immunomodulation among strains [24, 26]. Trypomastigotes disseminate via lymph and blood to other tissues being able to infect any nucleated cell, but with certain tropism to macrophages, muscle cells (cardiac, smooth, and skeletal), and nervous cells. The cycle is completed when another vector ingests infected blood [17].

4. Genetic diversity

Initially it was thought that T. cruzi was a highly clonal species that had experienced little genetic mixing during its evolution [27]. However, due to increasingly detailed studies of the structure of populations and nuclear and mitochondrial DNA of T. cruzi, it has been suggested that in addition to clonal propagation, there have been more recent and frequent hybridizations and genetic exchange than previously thought [28]. At present, there are six discrete typification units (DTU TcI-VI) accepted by international consensus [29] that include at least two hybrid lineages (TcV and TcVI) and an additional one mainly found in bats (TcBat) [30, 31], closely related to TcI. Among these DTUs, TcI is the most diverse and widely distributed lineage with the smallest genome, the least amount of aneuploidy, and probably related with some hybrid lineages [28]. The association of a particular T. cruzi strain with the diverse spectrum of the disease has not been completely established [32], although it has been possible to show some correlations. For example, Chagas disease megasyndrome is mainly found in South America where TcV and TcVI predominate. On the other hand, in North and Central America, cardiomyopathy is more common with Tc1 being strongly associated with human infection [33].

5. Chagas disease

In humans, Chagas disease progresses through two phases, acute and chronic, with the development of a symptomatic or an asymptomatic form [34]. The initial acute phase evolves within 1–2 weeks after the inoculation, lasts for 4–8 weeks, and is characterized for high levels of blood parasitemia. Affected individuals show moderate and unspecific symptoms such as fever, general discomfort, and hepatosplenomegaly. Sometimes it is possible to observe a cutaneous node (inoculation chagoma) or a unilateral bipalpebral edema (Romaña sign) that indicates the parasite inoculation site [34, 35]. The majority of acute infections are never detected. Only in less than 1% of the cases, encephalitis or myocarditis occurs that can be fatal mainly in children and the elderly [36]. The initial increase in parasitemia is contained by proinflammatory cytokines (IL-12, TNF-α, IFN-γ) and microbicidal substances synthesized by macrophages (reactive oxygen and nitrogen species) and NK cells (perforins). This initial innate immune response is followed by the development of an acquired immune response characterized by a polyclonal lymphocyte activation against the parasite mediated by T CD4⁺, CD8⁺, and B lymphocytes, which together reduce the parasite load, but do not completely eliminate T. cruzi, which still survives in the host tissues, thus initiating the chronic phase [37, 38]. Observations from research conducted before 1990 show that after being in the acute phase for 10–30 years, 30–40% of patients will turn to the symptomatic chronic phase and present digestive and/or cardiac compromise, while the rest will remain in an indeterminate phase for the rest of their lives [39, 40, 41]. Recent studies performed in children and teenagers from endemic areas in Mexico suggest that the time for the outcome of the CCC symptoms is shorter, months in some cases. Therefore, more research is required to clarify this aspect [42, 43].

5.1 Gastronitestinal form of Chagas disease

The gastrointestinal form of Chagas disease affects the whole digestive tract, with the esophagus and colon being the most altered. They present anatomic and motor disturbances as a result of chronic inflammatory lesions, focal myositis, fibrosis, and damage to intramural neurons [44], which in the esophagus could lead to dysphagia, odynophagia, epigastric or retrosternal pain, cough, and regurgitation. In advanced stages, the denervated esophagus is unable to transport the bolus, retaining it at the level of the cardia, which can cause weight loss, malnutrition, and repetitive aspiration pneumonitis [46]. On the other hand, the megacolon is characterized by prolonged constipation that could lead to fecaloma, abdominal distension, and intestinal obstruction. Despite treatment with laxatives, patients worsen and may suffer from volvulus due to intestine torsion [45, 47].

5.2 Chagasic chronic cardiomyopathy

The chronic chagasic cardiomyopathy is the most critical clinical manifestation of Chagas disease due to its high morbidity and mortality in endemic regions [48, 49]. It is characterized by a complex pathogenesis, and many aspects are still under investigation. The causes for the colossal cardiac damage experienced by CCC patients have not been thoroughly explained, and different theories have been exposed throughout the years. It was previously thought that the cardiac damage was only caused by the direct action of the parasite over cardiomyocytes; however, it is now known that there are other contributing factors such as inflammatory response, autoimmunity, microvascular abnormalities, and nervous damage [50]. The cardiac tissue damage is progressive and characterized by chronic inflammation, myocytolysis, and fibrosis [51]. A recent study revealed that during a T. cruzi infection, activated macrophages release the metalloproteinases MMP2 and MMP9 that activate TGF-β signaling for cardiac extracellular remodeling and thus fibroblast differentiation to myofibroblasts that is a cellular phenotype present during damage and possesses characteristics that make it suitable for healing functions [51].

The most critical heart lesions are located in the myocardium and can lead to heart failure, arrhythmias, and thromboembolism. The heart excite-conducting system is also affected, blocking completely or incompletely some of the branches of the bundle of His (mainly the right) and sometimes a complete blockade of the atrioventricular node [50, 52].

The number of parasites in the cardiac tissue of CCC patients is scarce. This, together with the finding of immunoglobulins with affinity to muscarinic receptors and β-1 adrenergic expressed in the cardiomyocyte surface and the appearance of cytotoxic T lymphocytes with reactivity toward myocardial fibers, suggested that the etiology of CCC was autoimmune [53, 54, 55, 56, 57]. Investigations performed during the last 30 years in animal models infected with T. cruzi and patients with Chagas disease have modified this theory. Nowadays it is known that the amount of T. cruzi antigens correlates with the intensity of the inflammatory infiltrate that acts against the parasites that reside in the tissue [58]. Such infiltrate is mainly composed of macrophages and CD8⁺ and CD4⁺ T lymphocytes (2:1 ratio) that show a Th1 cytokine profile (TNF-α, IFN-γ, IL-1, IL-2) [59, 60, 61]. At first, this Th1 response protects the host, but its exacerbation produces diffuse myocarditis that over time causes myocytolysis and reparative fibrosis with interstitial deposition of collagen fibers, whose progression is directly correlated with cardiac dilatation and deterioration of systolic function. The growth of the left ventricle is common, although it is also possible to observe it in the right ventricle and auricles [52, 62]. In advanced stages of the disease, it is possible to find a cardiac apical aneurism, pathognomonic of CCC [49]. Studies carried out in patients and postmortem analysis have demonstrated the presence of intracavitary thrombi accompanied by infarcts in several organs such as the lungs, kidneys, and brain [63, 64, 65].

At present, the factors that contribute to the progression of patients from the indeterminate phase to the determinate phase of Chagas disease have not been fully elucidated. As for other infectious diseases, the prognosis of CD depends on factors attributable to the host and the pathogen. Being an intracellular parasite, T. cruzi needs to modulate the defense mechanisms of the host cell in order to guarantee its survival. One of these mechanisms is apoptosis.

6. Apoptosis

The word apoptosis has its etymological origin in the Greek apó, which means “from” and ptosis, which means “falling off.” The term encompasses a genetically regulated process of cell death through which a cell destroys itself [66] with the key participation of cysteine-dependent proteases called caspases that are specific for aspartic acid [67]. Caspases are functionally divided in initiator (caspases 8, 9, and 10) and executioner (caspases 3, 6, and 7) [68]. In metazoans, apoptosis is an essential step in a great variety of physiological events such as embryogenesis, tissue remodeling, and the elimination of damaged or non-functional cells [69].

The sequence of events that lead to apoptosis can be unchained by two main routes: the extrinsic and the intrinsic pathways [66]. The intrinsic pathway initiates with the binding of death ligands (TNF-α, Fas-L, among others), present in soluble form or in the surface of effector cells, to their respective death receptors localized in the membrane of target cells. This binding results in the recruitment of cytosolic factors into the cytoplasmic domains of the death receptor, forming the death-inducing signaling complex or DISC. Initiator caspases such as procaspase 8, 10, or both are recruited to the DISC where they are activated. Caspases 8 and 10 in turn activate the following caspases in the pathway [70].

On the other hand, the intrinsic pathway, also known as the mitochondrial pathway of apoptosis, can be induced by various factors such as environmental stress or absence of growth factors. In this pathway, the formation of pores in the outer mitochondrial membrane allows the release of cytochrome-c into the cytosol, as well as other molecules such as endonuclease-G and Bcl-2 proteins. The interaction between cytochrome c, procaspase 9, and protease activating factor 1 or APAF 1 stimulates the formation of a heptameric complex known as apoptosome, which recruits procaspase 9 molecules, activating them. Caspase 9 molecules proceed to activate the following procaspases that execute the pathway, inducing apoptosis [66, 70].

Cells that die by apoptosis undergo characteristic morphological and biochemical changes that include a reduction in size, the collapse of the cytoskeleton, the disassembly of the nuclear envelope, and the fragmentation and condensation of chromatin. There are changes in the cell membrane composition and structure. Phosphatidylserine (PS) is translocated to the outer face of the membrane and protrusions are formed that finally break into membrane-enclosed fragments called apoptotic bodies. The mitochondrial membrane also changes with the formation of pores in the outer sheath that provokes the loss of the membrane potential of this organelle. These traits are used to quantitatively assess apoptosis [71, 72]. In metazoans, apoptotic cells and cell fragments are rapidly recognized and phagocytosed by cells of the immune system such as macrophages, which efficiently recognize PS expressed on the membrane of apoptotic cells. This early removal of cellular debris prevents the inflammatory response [73].

7. Apoptosis-like death in T. cruzi

It may seem odd to think that single-celled organisms can undergo apoptosis themselves, but it is believed that this has benefits for the survival of the population and therefore of the species. Shortly after apoptosis was described in metazoans, Docampo et al., using transmission electron microscopy, observed cytoplasmic and nuclear features suggestive of apoptosis in epimastigotes of T. cruzi treated with β-lapachone, an o-naphthoquinone that inhibits the synthesis of DNA. Such alterations included plasma membrane blebbing, chromatin condensation, and mitochondrial membrane alterations [74]. In addition to Trypanosoma, apoptotic death has been described in other protozoan taxa such as Plasmodium, and Leishmania that show distinctive characteristics of apoptosis similar to those described in multicellular organisms; however, the mechanisms involved are not fully understood [75]. Since then, other research groups have reported the appearance of stress- and drug-induced apoptotic traits (DNA fragmentation, PS externalization, loss of mitochondrial membrane potential, and cytochrome-c release) in T. cruzi [76, 77] with phenotypic similarities between metazoan cell apoptosis and T. cruzi cell death, but also with important differences in the processes and molecules that participate in them. Although PS translocation is a remarkable apoptotic trait in mammalian cells, in T. cruzi seems to be a parasite strategy to counteract macrophage activation [78]. On the other hand, caspases, key participants in apoptosis, are not present in T. cruzi. Nevertheless, its genome contains the TcMCA3 and TcMCA5 genes that code for metacaspases, cysteine proteases structurally similar to caspases but, unlike the latter, they have specificity for basic amino acid residues and are dependent on millimolar concentrations of calcium [79, 80]. The TcMCA3 protein is expressed in the four main stages of the parasite (epimastigotes, metacyclic trypomastigotes, blood trypomastigotes, and amastigotes), while TcMCA 5 is only expressed in epimastigotes. Analysis performed by immunofluorescence has shown that the treatment of T. cruzi with human serum, to induce programmed cell death, provokes a change in the subcellular localization of both metacaspases translocating them to the nucleus [81]. It has been observed that the increase in the expression of TcMCA5 augments the sensitivity of epimastigotes to programmed cell death as compared with parasites that express it at physiological levels. For its part, TcMCA3 protects epimastigotes from natural cell death and seems to play an important role in the process of differentiation to infectious metacyclic trypomastigotes [81].

Another molecule that could play an important role in the regulation of apoptotic cell death in T. cruzi is the elongation factor-1 (EF-1). This molecule, usually found in the nucleus and cytoplasm of eukaryotic cells, is formed of two subunits (EF-1α and EF-βγδ), and plays an important role in protein synthesis and in processes such as mitosis and cell proliferation [82]. Using anti-TcEF-1α antibodies coupled with fluorescein isothiocyanate, it was possible to observe that TcEF-1α accumulates in the nucleus of T. cruzi epimastigotes cultured for more than 13 days, which also showed apoptotic traits [83]. Subsequent investigations revealed that changes in the expression levels of EF-1α modify the rate of apoptosis in a murine erythroleukemic cell line [84], which suggests that TcEF-1α could be a marker of apoptosis in T. cruzi. The similarities that have been found between mammalian apoptosis and the phenomenon observed in T. cruzi have led some researchers to call it “apoptosis-like cell death” [85]. Nevertheless, other authors have concluded that the type of death observed in Trypanosoma and other protozoan parasites does not have the characteristics of a regulated death, and it is more an incidental cell death or necrosis [86].

8. Apoptosis modulation by T. cruzi

8.1 Apoptosis induction

Being an obligate intracellular parasite, T. cruzi needs to modulate the immune response mechanisms of its host to complete its life cycle and guarantee its survival and propagation. One of these mechanisms is apoptosis that can be modulated by this parasite on several cell types such as lymphocytes, macrophages, and cardiomyocytes. Macrophages are one of the most important niches in the mammalian host for T. cruzi replication and they are crucial for the immune response against the parasite because, depending on the stimulus, can be classically or alternatively activated. Classically activated macrophages (M1) produce nitric oxide (NO) that has the ability to kill T. cruzi, whereas alternatively activated macrophages, belonging to the M2 spectrum, synthesize polyamines that participate in parasite proliferation [87, 88]. Thus, one of the most important mechanisms of protective immunity against T. cruzi is a Th1-type immune response mediated by CD4⁺ and CD8⁺ T lymphocytes that produce IFN-γ, which in turn activates macrophages toward a classical phenotype for the control of parasitemia [89]. In response to this, T. cruzi displays outstanding strategies to control the activation of macrophages and inhibit apoptosis such as a reduction in the production of toxic molecules, including NO and its derivatives [90, 91], and the escape from the parasitophorous vacuole [92]. One of the molecules involved in the interference with NO is phosphatidylserine (PS). Experimental evidence indicates that PS exposure is connected to the survival and reproduction of obligate intracellular parasites by inhibiting NO production from macrophages [93, 94]. This strategy has been demonstrated in the infection of murine macrophages activated with IFN-γ and LPS with T. cruzi where trypomastigotes expose PS in their membrane. PS expression promotes parasite engulfment by phagocytic cells, a significant decrease in the expression of NOS2, and an increase in the production of the anti-inflammatory cytokine TGF-β by the infected macrophages. This suggests that the exposure of PS by T. cruzi (an apoptotic trait) could be responsible for the induction of the anti-inflammatory response similar to that induced by apoptotic cells [78].

In addition to the exposure of PS in the surface of T. cruzi, other membrane components participate in the immune evasion strategies and are considered virulence factors. All stages of T. cruzi have in their plasma membrane glycoconjugates attached to the membrane via glycosylphosphatidylinositol (GPI) anchors such as GIPLs and GPI-anchored glycoproteins [95], transialidase, mucins, mucin-associated proteins, and gp63 metalloproteases. It has been shown that GIPLs, in the presence of IFN-γ, induce apoptosis in macrophages through the ceramide portion by a NO production-independent pathway [96]. Also, it has been revealed that the sialylation of GPI-mucins protects trypomastigotes from lytic antibodies and, most likely, from the action of complement [97]. The alpha galactosylceramide expressed by T. cruzi induces anergy in NK cells and an increase in IL-33 [5]. On the other hand, the trans-sialidase (TS) of T. cruzi is an enzyme that transfers sialic acid from glycoconjugates present in mammalian cells to the parasite surface favoring its pathogenesis due to the formation of adhesive and protective structures on its surface. Also, TS has been shown to induce apoptosis of cells of the immune system [96]. In the infections with T. cruzi, TS can be located on the surface of the parasite or it can be secreted away from the site of infection [98]. Experiments performed in mice where recombinant TS was administered showed that the enzyme is capable of inducing apoptosis of the thymus. Contrarily, mice treated with anti-TS neutralizing antibodies did not show abnormalities in the organ [99, 100]. Later on, these observations were corroborated with the TUNEL assay and found that apoptosis was activated by sialylation of the CD43 mucin, which is constitutively expressed on the surface of T lymphocytes and monocytes [97]. This effect was not observed in mice that were given lactitol, an inhibitor of the transferase activity of the enzyme [101].

The induction of apoptosis in macrophages infected with T. cruzi may represent a proliferative strategy. It has been shown that when macrophages are infected with T. cruzi and phagocytose apoptotic CD4⁺ T cells, there is an increase in parasite replication inside macrophages, which in turn undergo apoptosis releasing more infective forms of the parasite such as trypomastigotes and spheromastigotes [5]. As a response to infection, macrophages release TGF-β, IL-10, and PGE2, which together deactivate them, allowing the survival of the intracellular forms of the parasite, as has also been observed with Leishmania [102].

For decades, the immunological suppression that T. cruzi exerts on cells of the immune system such as CD4⁺ and CD8⁺ T lymphocytes has been studied in detail, both in murine models and in patients with Chagas disease. This suppression can be macrophage-dependent or independent. In the first case, the unfavorable environment for T CD4⁺ cell proliferation during the acute phase of T. cruzi infection is due to the production of IFN-γ and NO by activated macrophages. In the second case, the suppression of CD4⁺ is through the interaction of TCR and CD3. The elimination of these cells inhibits the production of IFN-γ, thus preventing classical macrophage activation and favoring the development of an M2 phenotype, which favors the persistence of the parasite [88].

Likewise, apoptosis triggers the release of anti-inflammatory cytokines such as IL-10 and TGF-β by phagocytes, allowing the parasite to survive and continue with the infection.

It has been observed that in patients with chagasic cardiomyopathy there is no proliferation of peripheral blood mononuclear cells due to the activation of induced-apoptosis (AICD) by T. cruzi antigens. This has been complemented by the observation of a higher percentage of apoptotic cells in the hearts of patients with cardiac damage as compared with asymptomatic patients. Experimental evidence shows that the induction of apoptosis in CD4⁺ T cells occurs through the induction of Fas/FasL and the activation of the executioner caspases 3 and 8 [103]. The participation of Fas/Fas-L was verified by in vivo injection of anti-FasL antibodies, which blocked the induced apoptosis of CD8⁺ T lymphocytes, improving the Th1 response against the parasite. Interestingly, the blockade of TNF-α and TRAIL with antibodies did not have the same effect [104]. The increase in Fas-L has also been observed in the serum of patients in a chronic phase with CCC symptoms indicating that apoptosis also takes place during this phase [105]. Patients with chagasic cardiomyopathy showed a reduction in the proliferative response of T lymphocytes, in addition to a high production of CD4⁺CD62L⁻ T cells and an increase in the intracellular production of TNF-α, as well as the expression of genes of the TNF/TNFR superfamilies and caspases [106].

8.2 Apoptosis inhibition

T. cruzi is also capable of inhibiting apoptosis in infected cells. In addition to macrophages and T cells, the parasite also infects cardiomyocytes. In vitro experiments performed on murine cardiomyocytes incubated with T. cruzi or cruzipain (a parasite cysteine protease) showed that both trypomastigotes and cruzipain promote cardiomyocyte survival when cultured in media containing minimal serum concentrations. This phenomenon was associated with increased phosphorylation of Akt kinase and increased expression of the antiapoptotic protein Bcl-2. Furthermore, cultures that were treated with cruzipain showed less caspase-3 activation despite serum deprivation, suggesting that this enzyme might be responsible for the antiapoptotic effect [107]. Additionally, Chuenkova and Pereira observed that Schwann cells infected with T. cruzi trypomastigotes are capable of surviving the proapoptotic stimulus induced by TNF-α, TGF-β, and H₂O₂. They found that the neurotrophic factor derived from the parasite (PNDF, a GPI-anchored neuraminidase and TS) interacts with the Akt kinase, increasing the expression levels of this protein and inhibiting the expression of at least 3 genes encoding proapoptotic proteins such as Bax, caspase-9, and the FOXO transcription factor, which together promoted cell viability [108].

It has been recently shown that T. cruzi amastigotes induce apoptosis in cardiomyocytes due to overexpression of Bax and reduced expression of Bcl-2 linked to trypomastigotes and amastigotes, respectively. The transcription factor STAT3, but not STAT1, was found to be active in cardiomyocytes due to trypomastigote infection. In addition, the TLR7 gene was observed to be overexpressed in cardiomyocytes incubated with trypomastigotes, which indicates that the TLR receptor is involved in intracellular recognition [109].

8.3 Apoptosis modulation by T. cruzi in other tissues

In addition to the modulation of apoptosis by T. cruzi of its main host cells, similar effects have been observed in other cell types. It has been shown that during the infection with T. cruzi, the parasite is able to induce apoptosis in human chorionic villi [110]. Also, the parasite has been shown to have an antiproliferative activity on the malignant melanoma cell line B16-BL6 [111].

The coinfection of T. cruzi with HIV can cause meningoencephalitis since both pathogens infect astrocytes. Interestingly, infected astrocytes with T. cruzi or T. cruzi and HIV, but not with HIV alone, showed a decrease in cell death and IL-6 secretion, which has a protective effect against death. This suggests that T. cruzi can reduce the induction of cell death by HIV and even more affect the replication of the virus [112].

9. Conclusion

Numerous studies have been performed with the aim to understand the molecular basis of the survival of T. cruzi in its host. T. cruzi is an intracellular parasite that needs to surpass different defense mechanisms and a hostile environment to persist inside host cells. To achieve this goal, this protozoan has developed a wide array of strategies that target different mechanisms and molecules in the host. One of the mechanisms that T. cruzi modulates is apoptosis. It has been shown that the parasite has the ability to induce or inhibit apoptosis of different cells. The profound knowledge of these targets will allow the development of strategies capable of interfering with the development of the disease. It remains to be elucidated which signal transduction pathway or pathways are responsible for establishing this relationship.

Acknowledgments

Dr. Fiordaliso Carolina Román Carraro is a recipient of a postdoctoral fellowship from Dirección General de Asuntos del Personal Académico, UNAM. M. in Sci. Diego Maurizio Coria Paredes is a Ph.D. student of Posgrado en Ciencias Biológicas, Facultad de Medicina, UNAM. This work was supported by grant number IN214522 from Papiit, DGAPA, UNAM and funding from División de Investigación, Facultad de Medicina, UNAM TO LGK.

Conflict of interest

The authors declare no conflict of interest.