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Keywords:

  • cytokines;
  • immune response;
  • myocarditis;
  • parasite persistence;
  • Trypanosoma cruzi

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Historic remarks
  5. Acute immune response in the myocardium
  6. Immune evasion strategies of Trypanosoma cruzi
  7. Mechanisms of chagasic cardiomyopathy: parasite persistence and autoimmunity
  8. The chronic extracellular matrix remodelling
  9. Concluding remarks
  10. Disclosures
  11. References

Chagas disease (CD) is caused by the infection with the protozoan haemoflagellate Trypanosoma cruzi. This disease is still a great menace to public health, and is largely neglected as it affects mostly the poorest populations of Latin America. Nonetheless, there are neither effective diagnostic markers nor therapeutic options to accurately detect and efficiently cure this chronic infection. In spite of the great advances in the knowledge of the biology of natural transmission, as well as the immunobiology of the host–parasite interaction, the understanding of the pathogenesis of CD remains largely elusive. In the recent decades, a controversy in the research community has developed about the relevance of parasite persistence or autoimmune phenomena in the development of chronic cardiac pathology. One of the most notable aspects of chronic CD is the progressive deterioration of cardiac function, derived mostly from structural derangement, as a consequence of the intense inflammatory process. Here we review the evidence supporting the multifactorial nature of Chagas heart disease comprising pathogen persistence and altered host immunoregulatory mechanisms.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Historic remarks
  5. Acute immune response in the myocardium
  6. Immune evasion strategies of Trypanosoma cruzi
  7. Mechanisms of chagasic cardiomyopathy: parasite persistence and autoimmunity
  8. The chronic extracellular matrix remodelling
  9. Concluding remarks
  10. Disclosures
  11. References

Trypanosoma cruzi is an intracellular protozoan which causes Chagas disease (CD). Endemic to several regions in Latin America, this disease persists as the major infectious heart disease in the world (1,2). It is estimated that around 75 million people live in risk areas (3) and 13 million people are currently infected in Central and South America. The global incidence of the disease is considered to be 300 000 new cases per year.

Natural transmission of the disease occurs through faeces of the vector (a haematophagous bug belonging to the subfamily Triatominae, family Reduviidae), deposited near a skin lesion or mucosa (80–90%), via organ transplantation or blood transfusion (5–20%) or congenital transmission (0·5–8%) (4). Majority of infections occur during early childhood, and around 30% infected people develop chronic cardiac involvement, usually after decades of asymptomatic infection (5).

The interaction between human beings and vectors is favoured by several factors. For example, human invasion of the natural habitat where Triatominae insects are endemic, deforestation and even climatic phenomena can contribute to domestic adaptation of novel species of transmitter haematophages (6,7).

The South Cone Initiative against CD, carried out as an attempt to control the transmission of the disease by eradicating the insects from residential settings, has had an impact on the natural transmission of the disease. As a result, it is estimated that the local annual incidence has fallen from ∼800 000 new cases in the 1980s to ∼200 000 today, and in some of these countries the disease was declared to be ‘controlled’ (8,9).

The maintenance of this trend depends on continued surveillance and interventions where necessary. However, CD is still a serious menace to public health in various countries (10,11). In fact, CD has been considered as one of the most neglected infectious diseases (1,12,13) mostly due to deficiencies in epidemiologic control, the persistence of some endemic foci, sporadic disease outbreaks, transmission through transplantation or by oral route, along with the absence of diagnostic and prognostic markers and the limited efficacy of the anti-parasitic drugs.

As mentioned above, the currently available therapeutic options for CD are limited. Most of the therapeutic measures are aimed at treating the consequences of disease such as cardiac failure. Specific antimicrobial therapy [trypanocidal therapy (TT), which consists mainly of nitrofurans and imidazoles], when established early during the acute phase of CD, can prevent progression to the chronic phase in 50–70% of the cases (14). Although a study showed reduced parasitism, inflammation and conduction disturbances after benznidazole administration to mice chronically infected with T. cruzi (15), there is inconsistent clinical evidence supporting the use of TT in chronic chagasic cardiomyopathy (CCC) or in the indeterminate phase of the disease (16,17), mainly because most of the cases are detected at advanced stages of the disease, and because of the scarcity of the parasite at these stages. However, ongoing studies are exploring if TT does offer therapeutic advantage in chronic patients (18).

During the acute phase of the infection, an exacerbated immune response is commonly observed in the myocardium of infected individuals, leading to collateral damage which in extreme cases may lead to a systemic inflammatory response and death. At advanced stages of the disease, additional pathological changes appear, including the development of conduction disturbances, dysautonomia, cardiomegaly, fibrosis, thinning of the ventricular wall and microvascular damage. The main challenge in the understanding of immunopathology during T. cruzi-induced myocarditis is why, despite a robust immune response in the myocardium during the acute phase of infection, the parasite is not completely eliminated from tissues (19), being able to persist chronically.

Historic remarks

  1. Top of page
  2. Summary
  3. Introduction
  4. Historic remarks
  5. Acute immune response in the myocardium
  6. Immune evasion strategies of Trypanosoma cruzi
  7. Mechanisms of chagasic cardiomyopathy: parasite persistence and autoimmunity
  8. The chronic extracellular matrix remodelling
  9. Concluding remarks
  10. Disclosures
  11. References

Almost a century ago, Carlos Chagas described the general pathologic features of American trypanosomiasis, characterized its main clinical features and described the aetiological agent and major form of transmission. Such level of comprehension reached by a single researcher in the pathogenesis of a disease constitutes an exceptional event in the history of medicine.

By then, Chagas was established in Lassance and involved in governmental public health attempts to eradicate malaria and yellow fever from Rio de Janeiro, where both diseases were prevalent. While he was working there, a local railway engineer told him about the existence of a haematophagous bug which dropped from the ceilings of huts onto the faces of people while they were asleep. They were known as ‘barbeiros’ (barbers) or ‘chupança’ (kissing bugs). Chagas began to speculate that these bugs might act as hosts for a microorganism and identified in them a flagellate parasites. He soon demonstrated that this previously unrecognized parasite (later named Trypanosoma cruzi) was able to infect mammals (20).

Chagas searched for evidence of animal infection with this parasite and found it in the bloodstream of a domestic cat. Shortly afterwards, he was asked to see a 2-year-old girl named Berenice who owned the infected cat, and who fell ill with fever. He was not only able to demonstrate the same organism in her blood in the acute phase of the illness, but also noted that it was cleared as she recovered.

In the next few years he described the most important chronic features of the disease, including the cardiac, gastrointestinal and neurological manifestations, which were endemic in the region. In 1911, he described the congenital transmission of the infection. He received many honours and awards for this work (21).

After almost two decades of being forgotten, several remarkable discoveries were made on the epidemiology of CD. Of note, the Argentinean researcher Salvador Mazza suggested in 1936 that the disease could be acquired through transfusions (22), and then Dias and Pellegrino described in 1947 that the burden of the disease could be controlled by elimination of the vector using insecticides (23). However, it was only in the early 1990s that the real magnitude of the disease was recognized, leading to the formulation of public health policies to exclude infected blood donors and to eradicate the vector from the houses. As mentioned before, these strategies have proved to be extremely efficient in the control of disease burden. [For a detailed description on the history of CD, see Ref. (24).]

Acute immune response in the myocardium

  1. Top of page
  2. Summary
  3. Introduction
  4. Historic remarks
  5. Acute immune response in the myocardium
  6. Immune evasion strategies of Trypanosoma cruzi
  7. Mechanisms of chagasic cardiomyopathy: parasite persistence and autoimmunity
  8. The chronic extracellular matrix remodelling
  9. Concluding remarks
  10. Disclosures
  11. References

Currently, there is no doubt that chagasic cardiomyopathy (CC) is the result of an inflammatory process. Indeed, one of the pathologic hallmarks of CC is the presence of a large number of inflammatory cells in the myocardium. Such infiltration of immune cells can be a response to the cardiac tropism of the parasite, or as a consequence of altered immunological tolerance; this remains controversial but certainly depends on the genetic background of the host, as some people never develop heart disease despite infection.

After invading a mammalian host, the replication of T. cruzi occurs within the cytoplasm of different cell types including macrophages, fibroblasts, skeletal and heart muscle cells, neuronal and epithelial cells. Particularly, the parasite has a tropism to myocardial cells and forms nests, a pathological feature of the acute disease (Figure 1). Immediately after parasitism, the recruitment of leucocytes to the tissue is triggered. The migration of immune cells depends on the local production of cytokines and chemokines, as well as the upregulation of expression of their receptors and adhesion molecules (26,27). In addition, these inflammatory cells have to cross the extracellular matrix, in a process that is orchestrated by the production and activity of extracellular matrix metalloproteinases (MMP) (28). We recently demonstrated that T. cruzi induces increased MMP activities in the cardiac tissue, and that this increased activity is associated with cellular inflammation as well as mortality during the acute phase of the experimental infection (29).

image

Figure 1.  Immunopathogenesis of Chagas heart disease. The tissue damage in Chagas heart disease starts with multiplication of Trypanosoma cruzi which leads to cardiomyocyte destruction (I). This starts a specific immune response against the parasite but also involves the release of cell components leading to bystander activation, whereby immune responses are generated against host cellular components (I). This bystander activation and molecular mimicry between parasite and host antigens generate autoantibodies (II and III) and autoreactive T cells (IV). The autoantibodies can generate lesions mediating complement activity (II) and opzonizing tissue for macrophage activation (III). Autoreactive CD8+ T cells recognize self-antigens and destroy cardiac tissue (IV). Cytolytic T lymphocytes not only destroy infected cells but also contribute to the destruction of adjacent cells (VI). Tissue injury leads to TNF-α production, resulting in nitric oxide production. Trypanosoma cruzi infection also induces nitric oxide production directly, which is responsible for the generation of extensive cardiac damage derived from oxidative stress (V). The immunopathological process continues during the chronic phase of infection. Despite lower parasite levels at this stage small and progressive intrafascicular inflammatory focuses continue to develop for years, ending in fibrosis and the development of dilated cardiomyopathy.

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Host resistance during experimental CD is dependent on both innate and acquired immunity, requiring the combined effects of a number of cell types, including NK cells (30), CD8+ (31,32) and CD4+ (33) T cells, as well as antibodies produced by B cells (35).

Cytokines play key roles in regulating both parasite replication and immune responses in infected animals. It has been demonstrated that the cytokines IFN-γ and TNF-α are involved in the protective response to T. cruzi (36–40). IFN-γ is synthesized shortly after infection, mainly by NK cells, in response to IL-12 and TNF-α (41). However, the CD4+ and CD8+ T cells also produce IFN-γ during T. cruzi infection (34). In concert with TNF-α, IFN-γ leads to the activation of inducible nitric oxide synthase (iNOS) (40,42), the enzyme that catalyses nitric oxide (NO) synthesis by macrophages and inhibits parasite replication(40,43).

A predominant Th1 reaction is central to the control of T. cruzi. The treatment of T. cruzi-infected mice with an mAb to IFN-γ results in exacerbation of parasitaemia and mortality (44–46). In contrast, the regulatory cytokines IL-4, IL-10 and TGF-β have been associated with susceptibility to T. cruzi infection by inhibiting the effects of IFN-γ and macrophage activation (47,48).

Among the factors characterizing the immune response elicited by T. cruzi infection, it has been described that chagasic patients have increased frequencies of activated lymphocytes in the blood (49). Trypanosoma cruzi-activated leucocytes produce large amounts of IFN-γ, IL-12 and TNF-α (42,47,48,50), as well as NO (51) and extracellular MMP (29). Additionally, they exhibit low production of anti-inflammatory cytokines like IL-4 or IL-10 (52,53). It is noteworthy that overproduction of cytokines and enhanced expression of chemokine receptors induce a massive migration of inflammatory cells to the myocardium and other organs (27,36,53–56). From these data, it is possible to infer that a host imbalance in immune response, along with the parasite’s ability to trigger a polyclonal immune stimulation, does play a central role in the maintenance of such persistent lymphocyte activation (57,58).

In addition, the acute and chronic phases of T. cruzi infection are characterized by extensive polyclonal activation of T and B lymphocytes (59). Several studies have demonstrated that CD8+ cells are a predominant cell population in the cardiac tissue of chronic chagasic patients (60–62). Besides their cytolytic activities, CD8+ T cells also contribute to immune responses to T. cruzi and immunopathogenesis (63) by producing cytokines such as IFN-γ and IL-10 (50,64).

Regulatory T cells

The recent identification of CD4+CD25+Foxp3+ regulatory T cells (Treg) as a natural mechanism of control of the immune response has stimulated research interest and the development of therapeutic strategies for autoimmune and inflammatory diseases. The presence of Tregs in an inflammatory site is generally regarded as contributing to pathogen persistence, because it maintains an attenuated immune response against infectious agents (65).

These cells modulate the immune response, suppressing the proliferation of co-cultured CD4+CD25 T cells and inhibiting both induction and effector functions of autoreactive T cells (65) through co-inhibitory molecules cytotoxic T-lymphocyte antigen 4 (CTLA-4) and cytokines (IL-10, TGF-β, IL-35) (66,67). TGF-β has been implicated as a mediator of immunosuppression, inhibiting IL-2 receptor induction (68), thymocyte proliferation (69), B-cell proliferation and differentiation (70), IFN-γ-induced class II antigen expression (71), cytotoxic lymphocyte generation and lymphokine activated killer cells (72).

Recent studies suggest that Tregs may not be important in the modulation of the immune response during experimental T. cruzi infection. Despite migrating to the myocardium of the infected host, they may not participate actively in the control of the inflammatory response, as the blockade of the high-affinity IL-2 receptor CD25 (one of the molecular markers of Treg) did not significantly alter the local or systemic inflammatory response (73–75).

However, it has recently been demonstrated that the approach of blocking CD25 is not sufficient to rule out a role for Treg cells. In fact, we confirmed a limited role for CD25 signalling in controlling the inflammatory response during this protozoan infection. However, our data also suggest that agonistic signalling through glucocorticoid-induced tumour necrosis factor receptor (GITR, which regulates Treg activities) induces increased heart inflammation, parasite replication and host resistance against the infection. These data suggest that Treg cells are involved in the immunological balance during T. cruzi infection (74). Tregs are also able to migrate to the site of cardiac inflammatory lesions triggered by T. cruzi, and can suppress effector function of CD4+ and CD8+ T cells during infection (73,75,76). In addition, the cytokine TGF-β, which is produced by these cells, is involved in the pathogenesis of collagen deposition and fibrosis in the chronic phase of CD (77). Thus, similar to other parasitic agents (65), T. cruzi may evade the immune system and persist in host tissues by inducing an endogenous population of Tregs or exploiting host immunoregulatory mechanisms.

Th17 cells

For years, immune responses in inflammation were explained based on a dichotomy of cytokines that are produced. However, the Th1–Th2 paradigm has been reconsidered, as a novel lineage of effector CD4+ T helper lymphocytes, Th17, which produces IL-17A and IL-17F, IL-21, IL-22 and TNF-α, has been described (78). Differentiation of naïve T cells into Th17 may be mediated by the combined effect of the transcriptional factors RORγt and RORα, which are dependent on activation of the signal transducer and activator of transcription STAT-3 and require the cytokines IL-1β, IL-6, IL-21, TGF-β, as well as the expression of the chemokine receptor CCR6 (79,80). IL-17 has pro-inflammatory properties and acts by inducing fibroblasts, endothelial cells, macrophages and epithelial cells to produce a range of inflammatory mediators, such as granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-1, IL-6, TNF-α, iNOS, metalloproteinases and chemokines (CXCL1, CXCL2, CXCL8, CXCL10), leading to the recruitment of neutrophils and inflammation (81–83).

T-helper-17 cells have been linked to the pathogenesis of several inflammatory and autoimmune diseases such as multiple sclerosis, psoriasis, rheumatoid arthritis, colitis, autoimmune encephalitis (84) and some infectious diseases including schistosomiasis (85), toxoplasmosis and tuberculosis (86). Beyond Th17 cells, IL-17 is also produced by other cells types including CD8+ T cells, γδ T cells, neutrophils, monocytes and NK cells (87).

We have recently evaluated the role of IL-17 during T. cruzi infection. Trypanosoma cruzi-infected mice were treated with anti-mouse IL-17 mAb, which resulted in premature mortality, reduction in cardiac parasitism, enhanced production of TNF-α, IFN-γ, chemokine and chemokine receptors, expression of type 1 response and increase in cardiac inflammatory infiltrates compared with control animals (Guedes P.M.M., unpublished data). Our results indicate that IL-17 may be important in the control of cardiac inflammation by playing a negative feedback role on production of TNF-α, IFN-γ and chemokines during experimental T. cruzi infection, modulating the cardiac immune pathology of CD.

Chemokines

Chemokines also play important roles in immune modulation, leucocyte activation, co-stimulation and differentiation during innate and adaptive immune responses (88–90). Human and murine macrophages as well as cardiomyocytes produce the chemokines CCL-2, CCL-3 and CCL-5 after being infected with T. cruzi and respond to these chemokines in vitro (91) by increasing T. cruzi uptake, enhancing NO production and controlling parasite replication (56,92). The cytokines IFN-γ and TNF-α are essential for the production of CCL-5 and CCL-3, respectively (36).

Thus, it is possible that IFN-γ-induced chemokines produced in the cardiac tissue of T. cruzi-infected individuals could also participate in the control of parasitism. Indeed, one study has demonstrated the differential co-expression of the chemokine receptors (CCR2, CCR3, CCR5, CXCR3 and CXCR4) and intracellular cytokines (IL-4, IL-10, TNF-α and IFN-γ) on CD4+ and CD8+ peripheral T cells from individuals with indeterminate (IND) or cardiac (CARD) clinical forms of CD after in vitro stimulation with T. cruzi antigens(53).

In addition, it has been observed that the percentage of CD4+ and CD8+ T cells co-expressing CCR5 and IFN-γ, CXCR3 and IFN-γ, and CXCR3 and TNF-α are higher in individuals classified with the cardiac than that with the indeterminate form of the disease. On the other hand, the percentage of CD4+ or CD8+ T cells co-expressing CCR3 and IL-10 or co-expressing CCR3 and IL-4 were lower in CARD individuals than in IND individuals (53). CCR5 and CXCR3 are important immunological markers of Th1 responses, while CCR3 and CCR4 are associated with Th2 responses (93). These results indicate that a T. cruzi-exacerbated specific type 1 immune response developed by CARD chagasic patients is associated with the development of heart pathology.

The strong inflammatory activity of T. cruzi activated lymphocytes seems largely harmful rather than beneficial to the host. In order to minimize collateral damage from excessive inflammation, the initial migration of cells into the heart tissue should be controlled. However, the extent of this inflammatory response should also act to prevent parasite persistence (56). Thus, it is possible that in the absence of such adequate immune response regulation, the extent of myocardial inflammatory damage would be higher, as demonstrated by high levels of IFN-γ observed in patients with CC (50). Strategies targeting natural regulatory mechanisms in addition to antimicrobial treatment may be beneficial but have been not explored in research of chemotherapy against T. cruzi (94).

Cytokines

Various cell types and soluble molecules have been shown to participate in the control of infection as well as in the induction of pathogenesis during T. cruzi infection. Among them, the study of the profile of cytokines that are produced during infection may constitute a valuable key to the understanding of the immunopathological mechanisms involved in CC and controlling the immune response (54,95).

Some studies have demonstrated that peripheral lymphocytes as well as mononuclear cells infiltrating the heart tissue of patients with CC produce significantly more IFN-γ, TNF-α and IL-6, and less IL-4 and IL-10, than blood cells from infected asymptomatic individuals (50,96,97). In addition, peripheral mononuclear cells from patients with the indeterminate form produce high levels of IFN-γ and TNF-α associated with high IL-10 levels (53,97–100). On the other hand, other studies failed to demonstrate a correlation between production of inflammatory or anti-inflammatory cytokines and the clinical signs of CD (101). Thus, further studies are required to thoroughly establish if there is a correlation between the different cytokines present in cardiac tissue, lymphocyte culture or in serum during T. cruzi infection, with the outcome in terms of intensity of myocardial damage.

Immunoglobulins

During the sub-acute stages of experimental (102,103) and human (104) T. cruzi infection, the number of immunoglobulin-secreting cells in the spleen and peripheral lymph nodes is very high and the majority of activated B cells secrete nonspecific antibodies. These responses may lead to the potential expansion of self-specific clones that might be responsible for the killing of parasitized and nonparasitized cells.

In this phase of experimental mice infection, the B-cell polyclonal proliferation is characterized by a typical isotypic profile, IgG2a, and IgG2b in the spleen and lymph nodes (59). The molecular mimicry between parasite proteins and several self-antigens has been widely described. For example, autoantibodies against cardiac myosin, tubulin, actin, ribosomal P protein, keratin, β-adrenergic and muscarin receptors, myoglobulin, thyroglobulin, myelin and Cha antigen (105–109) have been detected in infected individuals. Recently, anti-beta (1)-adrenoceptor autoantibodies were found in sera of patients with idiopathic dilated cardiomyopathy. This finding could link the pathogenesis of chagasic dilated cardiomyopathy to the existence of these antibodies (110). However, the mechanisms implicated in the generation of such autoantibodies, as well as their role in cardiac pathology, remain to be established.

Antibodies are not only involved in the resistance to T. cruzi infection by opsonization, but may also mediate tissue destruction induced by complement (Figure 1). Several studies have indicated the importance of antibodies for host survival and parasite clearance (35,111,112). Indeed, mice deficient in B lymphocytes are hyper-susceptible to T. cruzi infection, demonstrating premature mortality rates and increasing parasitaemia (113).

The biological properties of IgG antibodies may be diverse, and have an important effect on the natural history of CD. The production of different IgG isotype subclasses is controlled by distinct cytokines. In chronic chagasic patients, IgG1 and IgG2 form about 90% of the IgG produced (114). Th1 cytokines (IL-12, IFN-γ, TNF-α) are responsible for the production of IgG1 and IgG3 isotype subclasses, while Th2 cytokines (IL-4 and IL-10) stimulate IgG2 production (115,116). IgG1 mediates lysis binding to complement C1q protein and macrophage phagocytosis, while IgG2 mediates immunity by nonphagocytic effector cells (117).

It has not been clearly defined whether a correlation between the different clinical forms of CD and the levels of IgG isotype subclasses exists. Several authors have tried to demonstrate the correlation between IgG isotype subclasses and the severity of the different clinical forms of CD in patient groups (118–120) and in experimental models (102,121). While some studies detected no differences between immunoglobulin levels among individuals with different clinical manifestations (118,120), others observed higher levels of IgG2 antibodies in the sera of patients with cardiac and gastrointestinal manifestations of disease (119,122).

Immune evasion strategies of Trypanosoma cruzi

  1. Top of page
  2. Summary
  3. Introduction
  4. Historic remarks
  5. Acute immune response in the myocardium
  6. Immune evasion strategies of Trypanosoma cruzi
  7. Mechanisms of chagasic cardiomyopathy: parasite persistence and autoimmunity
  8. The chronic extracellular matrix remodelling
  9. Concluding remarks
  10. Disclosures
  11. References

Trypanosoma cruzi has several strategies to evade the immune system, via induction of TGF-β production by immune cells, induction of lymphocyte apoptosis, transfer of sialidase to the cellular membrane, antigenic competition, acquired tolerance, as well as the possible blocking role of soluble antigens or circulating immune complexes. The release of excretory–secretory products which have potent immunosuppressive activity is a direct evasion mechanism.

Apoptosis in immune cells can be induced by factors such as the CD95 receptor ligand system (Fas–Fas-L) (123) and by cytokines such as TNF-α and IFN-γ (124). Trypanosoma cruzi induces apoptosis of lymphocytes by several mechanisms. For example, the high levels of NO induced in vitro and in vivo by T. cruzi lead to apoptotic cell death in many different cell types (125–127).

We showed that the induction of apoptosis by Fas–Fas-L interaction in T. cruzi-infected mice is dependent on NO and IFN-γ (126). Another strategy used by T. cruzi to induce lymphocyte apoptosis is mediated by trans-sialidase (TS). This enzyme transfers sialic acid from the microenvironment to the parasite’s mucins which entirely cover the parasite surface (128). Sialylated mucins are involved in parasite protection against components of the complement cascade. TS from T. cruzi not only acts on the parasite surface but is also shed into the milieu, being detected in the blood during the infection. The enzyme persists in blood because of the presence of a C-terminal region named shed acute-phase antigen (SAPA) (129,130).

Trypanosoma cruzi strains that produce and secrete higher amounts of TS and induce 100% mortality in mice induce thymic involution and thymocyte depletion in infected mice (131). Natural or recombinant TS injected into naive mice induce apoptosis in the thymus and in lymph nodes (132–134). Trypanosoma cruzi TS induces polyclonal lymphocyte activation and subsequent hypergammaglobulinaemia. It is noteworthy, however, that this polyclonal lymphocytic response appears to be important for host survival, as sensitization of mice with small doses of T. cruzi TS renders the mice highly susceptible to T. cruzi infection (135).

Sialylated mucins also confer on the parasite the ability to specifically inhibit some components of the innate immune system, such as the complement-mediated lysis. Trypomastigote forms of T. cruzi are resistant to complement-mediated lysis in the absence of lytic antibodies, whereas epimastigotes are sensitive to lysis via the alternative complement pathway (136,137). It was demonstrated that antibodies to a 160-kDa protein correlate with the capacity of the sera to support complement-mediated lysis of trypomastigotes (138).

The T. cruzi gp160 restricts complement activation by binding the complement component C3b and inhibiting C3 convertase formation. The protein is anchored in the parasite membrane via a glycosyl phosphatidylinositol linkage, similar to the human complement regulatory protein, decay-accelerating factor (139). Moreover, epimastigotes transfected with a T. cruzi expression vector carrying the trypomastigote complement regulatory protein (GP160) acquired resistance to lysis (140). Trypanosoma cruzi calreticulin (TcCRT) was also shown to inhibit the human complement system when binding to the collagenous portion of C1q and to inhibit the generation of classical pathway convertases and membrane attack complexes. Moreover, antibodies against TcCRT inhibit the phagocytic activity of macrophages (141).

The surface of trypanosomatid parasites contains large amounts of glycosylphosphatidylinositol (GPI), which occurs either as GPI anchors for glycoproteins and polysaccharides or as free glycoinositolphospholipids (GIPLs) that contain the identical core structure of GPI (142). GPI-anchored mucin from the T. cruzi membrane is capable of binding to the macrophage cell surface, altering activation of human macrophage and dendritic cells (143). The administration of C10 antigen (mucin-like molecule from T. cruzi membrane) reduced the secretion of TNF-α and IL-I2 by macrophage in a dose-dependent manner (143).

By contrast, T. cruzi GPI induces inflammatory responses by Toll-like receptor (TLR) 4 activation and resistance to T. cruzi infection (144). TLRs function as pattern-recognition receptors in mammals and play an essential role in the recognition of microbial components (145). However, TLR2−/− mice infected with T. cruzi produce higher levels of pro-inflammatory cytokines and nitric oxide than wild-type mice. Thus, TLR2 has an important immunoregulatory role preventing excessive activation of innate immunity and uncontrolled production of pro-inflammatory cytokine (146,147), suggesting that recognition of T. cruzi via TLR2 may be related to immune evasion strategies of the parasite.

Mechanisms of chagasic cardiomyopathy: parasite persistence and autoimmunity

  1. Top of page
  2. Summary
  3. Introduction
  4. Historic remarks
  5. Acute immune response in the myocardium
  6. Immune evasion strategies of Trypanosoma cruzi
  7. Mechanisms of chagasic cardiomyopathy: parasite persistence and autoimmunity
  8. The chronic extracellular matrix remodelling
  9. Concluding remarks
  10. Disclosures
  11. References

The complexity of this parasitic disease is better comprehended when observed from an evolutionary viewpoint. Trypanosoma is a very versatile genus, as it is able to parasitize a wide variety of animals, from reptiles to mammals (148). Trypanosoma cruzi has been a constant threat to humans for millennia (149). Both parasite and host must have evolved together during this ancient relationship, otherwise, one of the species would be extinct.

Chagas heart disease is essentially an infectious myocarditis (150). During the acute phase of the infection, there is no doubt that the inflammatory response is directed towards the parasite, which is present in large amounts in the myocardium. The opinion of the authors is that the extension of this acute damage to myocardial tissue, as well as the quality of the immune regulatory response, is related to the presence and extent of chronic cardiomyopathy. However, there are some individuals who develop an intense myocarditis but remain asymptomatic.

No evidence exists for an association between grade of acute myocarditis and different clinical outcomes, mainly because detection of individuals undergoing the acute phase is extremely rare. In addition, the experimental models fail to mimic the chronic phase of the infection (i.e. the infrequent development of dilated cardiomyopathy even in chronic-resistant mice strains). Thus, the study of the pathological basis of the chronic phase of the disease represents additional complexities that are derived from the nature and evolution of the immune response, the presence of the parasite and the host’s cardiac physiology.

From an immunologist’s perspective, the main controversial point in CD has been the specificity of the immune response raised in the myocardium. Two main hypotheses have been proposed, centred on two mechanisms wrongly proposed to be mutually exclusive: parasite persistence or autoimmunity (151). The authors believe that both these phenomena co-exist in CD. Thus, both immune evasion strategies of the parasite and immune homeostasis defects of the host that are determined mainly by genetic features are important for the development of the clinical forms of CD. Our opinion is that in the chronic phase of the disease, it is too limited to state that inflammation is the unique factor leading to pathogenesis. The healing and physiological adaptations of the heart after myocardial injury are crucial and determined by several host factors. The mechanisms of parasite persistence and the quality of the host immune response are both crucial in determining the extent of tissue damage (152).

The autoimmunity hypothesis arose from studies reporting that a strong inflammatory response can be found in advanced stages of the disease, even in the absence of parasites (153–155). Moreover, several experimental approaches unquestionably demonstrated the existence of autoreactive phenomena of cellular and humoral nature in infected individuals (108,155–158). Lymphocytes from infected mice and from chagasic patients are able to recognize self-epitopes (108,157). However, it has been difficult to demonstrate the pathophysiological relevance of this autoreactivity (159), as has been the case in several other autoimmune diseases triggered by infections.

The hypothesis of parasite persistence emerged as a potential mechanism to explain the chronic tissue damage following studies demonstrating genetic material from the pathogen in tissues of infected animals (160–163), where other techniques were not able to detect the presence of the microorganism (61,152). The parasite may also play an active role in the induction of cardiac pathology during CD (60,164). In fact, some evidence points a role for inherent parasite factors, such as biological and genetic variability among T. cruzi populations, in the different outcomes of the disease (165–167). A positive correlation between inflammation and the presence of T. cruzi in the myocardium, as well as the absence of T. cruzi in preserved areas of cardiac tissue, constitutes additional evidence that T. cruzi is able to induce myocardial damage directly (164,168).

Thus, the genetic characteristics of both host and microbe do determine not only the host resistance to the parasite itself, but also the adaptability of the host to the altered myocardial physiology, including the fibrotic response. Evolutionarily speaking, we may say that CD is the cost that humans have paid to survive T. cruzi.

The chronic extracellular matrix remodelling

  1. Top of page
  2. Summary
  3. Introduction
  4. Historic remarks
  5. Acute immune response in the myocardium
  6. Immune evasion strategies of Trypanosoma cruzi
  7. Mechanisms of chagasic cardiomyopathy: parasite persistence and autoimmunity
  8. The chronic extracellular matrix remodelling
  9. Concluding remarks
  10. Disclosures
  11. References

One of the most notable aspects of chronic CD is the progressive deterioration of cardiac function, derived mostly from structural derangement (169), as a consequence of the intense inflammatory process (Figure 1). This structural damage can be perpetuated by some factors derived from the parasite and from the host itself (54,170). Other disease features like dysautonomia (171) and microvascular derangements caused directly by the parasite can also add further complexity to the pathophysiology of CD.

Cardiac extracellular matrix (CECM) actively participates in the process of migration of inflammatory cells (28,172), as some of its components need to be transiently broken in order to establish the inflammatory infiltrate in the tissue. Matrix metalloproteinases are essential for the local rupture of CECM during inflammation and tissue repair. Nonetheless, MMP are also able to modify the molecular structure of soluble inflammatory mediators such as cytokines and chemokines, thus acting as modulators of their activities. These enzymes are particularly active in CECM in response to several stimuli, ranging from ischaemia to inflammation. Increased MMP activity (mostly of MMP9) has been reported during the progression of cardiac failure because of other aetiologies (28). While participation of MMPs in acute cardiac inflammation has been demonstrated (29), their role in remodelling of myocardial structure during the chronic phase of the disease remains an avenue for investigation.

Concluding remarks

  1. Top of page
  2. Summary
  3. Introduction
  4. Historic remarks
  5. Acute immune response in the myocardium
  6. Immune evasion strategies of Trypanosoma cruzi
  7. Mechanisms of chagasic cardiomyopathy: parasite persistence and autoimmunity
  8. The chronic extracellular matrix remodelling
  9. Concluding remarks
  10. Disclosures
  11. References

As in other complex pathologies, a global perspective on this disease is important to gain understanding of the role of some concepts in its pathophysiology. Thus, CD could be considered as the cumulative contribution of diverse mechanisms which vary in their intensity among individuals depending on their genetic background. These mechanisms are triggered immediately upon infection and involve all components of the immune response, the tissue regenerative response and cardiac physiological responses. The immunological mechanisms are the focus of this review. The tissue regenerative response involves mainly the extracellular matrix remodelling. Among cardiac physiological responses we would mention the electrophysiological dysfunctions which may be the consequence of parasite invasion of cardiac cells or due to collateral damage from free nitrogen and oxygen radical species.

In our opinion, the presence of the parasite within the myocardium is important in triggering and maintaining the immunopathogenic process during CD. However, we also believe that a unbalanced immune homeostasis can trigger parallel autoimmune phenomena which amplify the immune response, thus worsening the outcome of the disease.

Disclosures

  1. Top of page
  2. Summary
  3. Introduction
  4. Historic remarks
  5. Acute immune response in the myocardium
  6. Immune evasion strategies of Trypanosoma cruzi
  7. Mechanisms of chagasic cardiomyopathy: parasite persistence and autoimmunity
  8. The chronic extracellular matrix remodelling
  9. Concluding remarks
  10. Disclosures
  11. References

This work was supported by grants from: Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, grant number 05/60762-5 to FRSG and 2007/53940-0 to JSS), Centro Nacional de Desenvolvimento Cientifico e Tecnológico (The Millennium Institute for Vaccine Development and Technology grant number 420067/2005-1 to RTG and JSS and Doenças negligenciadas grant number 410467/2006-5 to JSS), and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Grant number 288/05-5 to PMMG).

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  7. Mechanisms of chagasic cardiomyopathy: parasite persistence and autoimmunity
  8. The chronic extracellular matrix remodelling
  9. Concluding remarks
  10. Disclosures
  11. References
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