**2.1.4 Molecular mimicry**

The induction of autoimmunity by similarities between *T. cruzi* and host epitopes has been long proposed as a mechanism that leads to tissue damage in the chronic phase of the disease. Both humoral and cellular autoimmune responses have been described, but we will discuss them in more detail in the section of immune system. The real importance of molecular mimicry in the pathogenesis of chagasic myocarditis is still a matter of debate (Girones, et al., 2005).

Although it seems that in some cases this mechanism triggers autoimmunity, in many others, autoimmunity seems to be an epiphenomenon of cellular destruction, with exposition of intracellular epitopes not normally exposed to the immune system. This, in turn may activate autoreactive lymphocytes leading to the appearance of autoantibodies that are not the cause of damage, rather a consequence (Girones, et al., 2005).

The most important cross reacting epitopes of *T. cruzi* and the correspondent epitopes in humans are listed in table 1, as well as the kind of immune response they elicit.

#### **2.2 Second ingredient: host immune system**

When the three ingredients theory was first proposed (Storino and Milei 1994, Milei, et al. 1996), second ingredients were mainly T lymphocytes and macrophages. In the subsequent years some evidence grew about the participation of humoral immune system through autoantibodies in the pathogenesis. As a consequence, the whole immune system of the host is now considered as the second ingredient.

As described earlier, mononuclear cells persist in the chronic stage of the disease, contributing to the inflammation through its products of secretion or through its own cytotoxicity (suppressor T cells) and cytolytic action (macrophages) (Storino & Milei, 1994).

As previously stated, molecular mimicry may be the main explanation of autoimmunity, triggering both cellular and humoral autoreactivity (Girones, et al., 2005). Figure 2 summarizes the most important immune events in CCC pathogenesis.

#### **2.2.1 Innate immunity**

In recent years innate immunity came to the attention of researchers of Chagas' disease pathogenesis. The role of NK cells has been particularly studied in early and late indeterminate forms of the disease and in CCC patients. In early indeterminate patients,

Once *T. cruzi* reaches blood torrent, it invades a great variety of cells in the host. When parasiting non phagocytic cells, *T. cruzi* uses some surface glycoproteins to attach to cell: gp82, gp30 and gp35/50. All three glycoproteins are known to induce calcium mobilization from intracellular reservoirs. Gp82 is linked to the phospholipase C (PLC) and inositol 1,4,5 – triphosphate (IP3). Gp 35/50 is associated to increasing intracellular levels of cyclic AMP. On the other side, cruzipain, a protein known to be secreted by *T. cruzi*, acts on kininogen and produces bradykinin, which binds to its receptor, further increasing intracellular calcium. Increased intracellular calcium produces modifications in cytoskeleton that lead to

In the parasitoforous vacuole, mainly by the action of gp85/TS a glycoprotein with transsialidase action, and TcTox, a protease, the parasite degrades the membrane of the vacuole,

The induction of autoimmunity by similarities between *T. cruzi* and host epitopes has been long proposed as a mechanism that leads to tissue damage in the chronic phase of the disease. Both humoral and cellular autoimmune responses have been described, but we will discuss them in more detail in the section of immune system. The real importance of molecular mimicry in the pathogenesis of chagasic myocarditis is still a matter of debate

Although it seems that in some cases this mechanism triggers autoimmunity, in many others, autoimmunity seems to be an epiphenomenon of cellular destruction, with exposition of intracellular epitopes not normally exposed to the immune system. This, in turn may activate autoreactive lymphocytes leading to the appearance of autoantibodies

The most important cross reacting epitopes of *T. cruzi* and the correspondent epitopes in

When the three ingredients theory was first proposed (Storino and Milei 1994, Milei, et al. 1996), second ingredients were mainly T lymphocytes and macrophages. In the subsequent years some evidence grew about the participation of humoral immune system through autoantibodies in the pathogenesis. As a consequence, the whole immune system of the host

As described earlier, mononuclear cells persist in the chronic stage of the disease, contributing to the inflammation through its products of secretion or through its own cytotoxicity (suppressor T cells) and cytolytic action (macrophages) (Storino & Milei, 1994). As previously stated, molecular mimicry may be the main explanation of autoimmunity, triggering both cellular and humoral autoreactivity (Girones, et al., 2005). Figure 2

In recent years innate immunity came to the attention of researchers of Chagas' disease pathogenesis. The role of NK cells has been particularly studied in early and late indeterminate forms of the disease and in CCC patients. In early indeterminate patients,

that are not the cause of damage, rather a consequence (Girones, et al., 2005).

summarizes the most important immune events in CCC pathogenesis.

humans are listed in table 1, as well as the kind of immune response they elicit.

**2.1.3 Cell host invasion and intracellular survival by** *Trypanosoma cruzi*

escapes from it and proliferates within the cell (Alves & Colli, 2007).

parasite endocytosis (Yoshida & Cortez, 2008).

**2.2 Second ingredient: host immune system** 

is now considered as the second ingredient.

**2.1.4 Molecular mimicry** 

(Girones, et al., 2005).

**2.2.1 Innate immunity** 


Table 1. Examples of cross-reacting epitopes (Girones, et al., 2005, Marin-Neto, et al., 2007).

compared to non infected people, increased values of pre-natural killer (NK)-cells (CD3- CD16+ CD56-), and higher values of proinflammatory monocytes (CD14+ CD16+ HLA-DR++) were found. The higher values of activated B lymphocytes (CD19+ CD23+) contrasted with impaired T cell activation, indicated by lower values of CD4+ CD38+ and CD4+ HLA-DR+ lymphocytes, a lower frequency of CD8+ CD38+ and CD8+ HLA-DR+ cells; a decreased frequency of CD4+ CD25HIGH regulatory T cells was also observed. All these data suggest a rather proinflammatory profile (Vitelli-Avelar, et al., 2006). This profile may be useful to limit parasitemia and confine infection to tissues. In fact, it has been demonstrated that NK cells are important in defense against the spread of parasitic infection (Brener & Gazzinelli, 1997), and are an important source of INF-a key cytokine to activate macrophages and help with parasite clearance (Camargo, et al., 1997).

In late indeterminate form, CD3-CD16+CD56+ and CD3-CD16+CD56DIM NK cells are increased but are in normal range in CCC patients, suggesting a protective role for them (Vitelli-Avelar, et al., 2005). NK cells showing CD56DIM may play a role in the down

Pathogenesis and Pathology of Chagas' Chronic Myocarditis 125

Monocytes from indeterminate patients, when infected *in vitro* with *T. cruzi*, express low levels of HLA-DR and high levels of CD80, a ligand for CTLA-4 (Souza P. E., et al., 2004). The interaction of these monocytes with CTLA-4+ T cells leads to the expression of IL-10, a cytokine known to down-modulate inflammatory responses (Gomes, et al., 2003). This is not observed in CCC patients. CD28- T cells, not expressing CTLA-4, express TNF- and INF-

In the same direction, CD4-CD8- T cells are found to be increased in indeterminate patients compared with CCC ones. These cells are also linked to the production of IL-10 and

Cells infiltrating myocardium have also been studied. As demonstrated with immunostaining of endomyocardial biopsies by our group, leukocytes infiltrating myocardium in Chagas' disease were approximately 50% macrophages, and 50% lymphocytes, mainly T lymphocytes (Milei, et al., 1992b). Further immunohistochemical characterization of these cells with CD45R for lymphocytes, CD20 and lambda and kappa light chains for B lymphocytes, CD45R0 for T lymphocytes and CD68 for macrophages,

Autoreactive T cells have caught the attention of many investigators. In experimental models, CD4+ T cells from infected mice showed a proliferative response to the exposition to human cardiac myosin heavy chain and to *T. cruzi* B13 protein. They also arrested the beating of fetal heart cells and, more importantly, induced myocarditis in immunized mice and promoted rejection of transplanted normal hearts in the absence of *T. cruzi* (Ribeiro-Dos-Santos, et al., 2001). Also, it has been described that T cells infiltrating the myocardium of chagasic patients cross react with human cardiac myosin heavy chain and to *T. cruzi* B13 protein and express high levels of INF- and low levels of IL-4, switching to a Th1 profile

In recent years, Treg cells have come to attention in relation to Chagas' disease pathogenesis. These cells are characterized by the expression of CD4, CD25 and FOXP3 (Ziegler & Buckner, 2009). Treg cells are increased in indeterminate patients compared to CCC, which correlates negatively with levels of activated CD8+ (Vitelli-Avelar, et al., 2005). A second subset of T CD4+ cells, recently described, the Th17 cells, resulted important in Chagas' disease pathogenesis. These cells, mainly linked to autoimmune pathology, are characterized by the expression of CD4+, RORt, and secrete IL-17 (Di Jin, et al., 2008). They were increased in a murine model of acute myocarditis induced by *T. cruzi* infection, as well as by immunization with heat-killed *T. cruzi* antigens (Bonney, et al., 2011). Both cell subsets seem to be related, as they require TGF- to differentiate. In the presence of proinflammatory cytokines, differentiation to Th17 cell prevails and a pro-autoimmune

An additional mechanism is the bystander activation. This is the activation of autoreactive lymphocytes by antigen presenting cells in a proinflammatory environment (Fujinami, et al., 2006). This kind of autoreactive T cells activation has been described in Chagas'disease

The importance of humoral immunity in controlling *T. cruzi* acute infection has been clearly established. Mice lacking B lymphocytes rapidly succumb to infection (Kumar & Tarleton,

1998). But the fact that attracted most attention is the production of autoantibodies.

a down modulator effect on inflammation (Villani, et al., 2010).

confirmed these findings (Milei, et al., 1996a).

(Cunha-Neto Edecio & Kalilf, 2001).

profile develops (Ziegler & Buckner, 2009).

**2.2.3 Humoral adaptative immunity** 

(Fedoseyeva, et al., 1999).

(Menezes, et al., 2004).

modulation of cytotoxic deleterious T CD8+ response reported in CCC patients (Sathler-Avelar, et al., 2009).

Monocytes display different cytokine profile. In indeterminate patients they produce more IL-10 (Gomes, et al., 2003) while in CCC patients they produce more TNF- (Vitelli-Avelar, et al., 2008), leading to a proinflammatory profile that could be responsible for chronic myocarditis.

Toll-like receptors (TLR) are also implied in the response to acute infection with *T. cruzi*. TLR-2 has been shown to recognize GPI surface molecules from the parasite. In vitro and in vivo studies have demonstrated that macrophages stimulated with GPIs through TLR-2/CD14 receptors produce NO, TNF- and IL-12 (Campos & Gazzinelli, 2004).

### **2.2.2 Cellular adaptative immunity**

The role of immune cells in the pathogenesis of Chagas' heart disease has been the dominant hypothesis for many years. The paucity of parasite cells in the inflamed myocardium and the presence throughout the evolution of the disease of macrophages and lymphocytes in patched infiltrates lead to this hypothesis. As early as in 1929, Magariños Torres, observing those infiltrates postulated an "allergic" mechanism for CCC. Further, Mazza and Jörg followed this thought and supported the "allergic" theory (Storino & Milei, 1994).

The study of circulating lymphocytes in peripheral blood of chagasic patients showed an increase in the percentages and actual numbers of double-positive cells of the phenotype CD3+/HLA-DR+, as well as decrease in the percentage of CD45RA+/CD4+ and CD45RA+/CD8+ T cells, indicating greater numbers of activated T cells circulating. Consistent parallel increases were seen also in the B lymphocyte subset which stained double-positive for CD19/CD5 (Dutra W. O., et al., 1994). These results were similar for both indeterminate and CCC patients. Moreover, activated T cells lacking the co-stimulatory molecule CD28 are increased in chagasic patients (Menezes, et al., 2004) and express high levels of HLA-DR molecules (Dutra, et al., 2000). Some interesting differences were demonstrated between indeterminate and CCC patients. CD28- T cells in indeterminate patients showed expression of CTLA-4, which recognizes the same ligands as CD28, but instead of inducing cell activation it causes down modulation of T cells. On the contrary, T cells in CCC patients do not up-regulate CTLA-4 (Souza P. E. A., et al., 2007). It is particularly interesting that CD8+CD28- cells are increased in CCC patients compared to indeterminate patients, and that these cells do not require co-stimulation to exert their cytotoxic functions. More strikingly, CD4+CD28- cells behave differently in indeterminate and CCC patients. In the formers, they are closely related to IL-10 levels, while in CCC patients they correlate with INF- levels (Menezes, et al., 2004).

Another interesting difference has been found in cellular response between indeterminate and CCC patients. CD4+ cells from CCC patients had an increased expression of V5+-TCR, not found in indeterminate patients. When CCC patients mononuclear cells from peripheral blood were cultured in the presence of trypomastigotes antigens, a selective expansion of CD4+ V5+ cells was obtained; while when cultured in the presence of epimastigotes antigens, an expansion of CD8+ V5+ cells was also noted. These findings could not be repeated in indeterminate patients. Trypomastigote stimulation led to the expansion of CD4+ V17+ in indeterminate patients, not seen in CCC patients. This suggests that CCC patients and indeterminate patients respond to different antigen repertoires (Costa, et al., 2000).

modulation of cytotoxic deleterious T CD8+ response reported in CCC patients (Sathler-

Monocytes display different cytokine profile. In indeterminate patients they produce more IL-10 (Gomes, et al., 2003) while in CCC patients they produce more TNF- (Vitelli-Avelar, et al., 2008), leading to a proinflammatory profile that could be responsible for chronic

Toll-like receptors (TLR) are also implied in the response to acute infection with *T. cruzi*. TLR-2 has been shown to recognize GPI surface molecules from the parasite. In vitro and in vivo studies have demonstrated that macrophages stimulated with GPIs through TLR-

The role of immune cells in the pathogenesis of Chagas' heart disease has been the dominant hypothesis for many years. The paucity of parasite cells in the inflamed myocardium and the presence throughout the evolution of the disease of macrophages and lymphocytes in patched infiltrates lead to this hypothesis. As early as in 1929, Magariños Torres, observing those infiltrates postulated an "allergic" mechanism for CCC. Further, Mazza and Jörg

The study of circulating lymphocytes in peripheral blood of chagasic patients showed an increase in the percentages and actual numbers of double-positive cells of the phenotype CD3+/HLA-DR+, as well as decrease in the percentage of CD45RA+/CD4+ and CD45RA+/CD8+ T cells, indicating greater numbers of activated T cells circulating. Consistent parallel increases were seen also in the B lymphocyte subset which stained double-positive for CD19/CD5 (Dutra W. O., et al., 1994). These results were similar for both indeterminate and CCC patients. Moreover, activated T cells lacking the co-stimulatory molecule CD28 are increased in chagasic patients (Menezes, et al., 2004) and express high levels of HLA-DR molecules (Dutra, et al., 2000). Some interesting differences were

patients showed expression of CTLA-4, which recognizes the same ligands as CD28, but instead of inducing cell activation it causes down modulation of T cells. On the contrary, T cells in CCC patients do not up-regulate CTLA-4 (Souza P. E. A., et al., 2007). It is particularly interesting that CD8+CD28- cells are increased in CCC patients compared to indeterminate patients, and that these cells do not require co-stimulation to exert their cytotoxic functions. More strikingly, CD4+CD28- cells behave differently in indeterminate and CCC patients. In the formers, they are closely related to IL-10 levels, while in CCC

Another interesting difference has been found in cellular response between indeterminate and CCC patients. CD4+ cells from CCC patients had an increased expression of V5+-TCR, not found in indeterminate patients. When CCC patients mononuclear cells from peripheral blood were cultured in the presence of trypomastigotes antigens, a selective expansion of CD4+ V5+ cells was obtained; while when cultured in the presence of epimastigotes antigens, an expansion of CD8+ V5+ cells was also noted. These findings could not be repeated in indeterminate patients. Trypomastigote stimulation led to the expansion of CD4+ V17+ in indeterminate patients, not seen in CCC patients. This suggests that CCC patients and indeterminate patients respond to different antigen repertoires (Costa, et al.,

T cells in indeterminate

2/CD14 receptors produce NO, TNF- and IL-12 (Campos & Gazzinelli, 2004).

followed this thought and supported the "allergic" theory (Storino & Milei, 1994).

demonstrated between indeterminate and CCC patients. CD28-

patients they correlate with INF- levels (Menezes, et al., 2004).

Avelar, et al., 2009).

**2.2.2 Cellular adaptative immunity** 

myocarditis.

2000).

Monocytes from indeterminate patients, when infected *in vitro* with *T. cruzi*, express low levels of HLA-DR and high levels of CD80, a ligand for CTLA-4 (Souza P. E., et al., 2004). The interaction of these monocytes with CTLA-4+ T cells leads to the expression of IL-10, a cytokine known to down-modulate inflammatory responses (Gomes, et al., 2003). This is not observed in CCC patients. CD28- T cells, not expressing CTLA-4, express TNF- and INF- (Menezes, et al., 2004).

In the same direction, CD4-CD8- T cells are found to be increased in indeterminate patients compared with CCC ones. These cells are also linked to the production of IL-10 and a down modulator effect on inflammation (Villani, et al., 2010).

Cells infiltrating myocardium have also been studied. As demonstrated with immunostaining of endomyocardial biopsies by our group, leukocytes infiltrating myocardium in Chagas' disease were approximately 50% macrophages, and 50% lymphocytes, mainly T lymphocytes (Milei, et al., 1992b). Further immunohistochemical characterization of these cells with CD45R for lymphocytes, CD20 and lambda and kappa light chains for B lymphocytes, CD45R0 for T lymphocytes and CD68 for macrophages, confirmed these findings (Milei, et al., 1996a).

Autoreactive T cells have caught the attention of many investigators. In experimental models, CD4+ T cells from infected mice showed a proliferative response to the exposition to human cardiac myosin heavy chain and to *T. cruzi* B13 protein. They also arrested the beating of fetal heart cells and, more importantly, induced myocarditis in immunized mice and promoted rejection of transplanted normal hearts in the absence of *T. cruzi* (Ribeiro-Dos-Santos, et al., 2001). Also, it has been described that T cells infiltrating the myocardium of chagasic patients cross react with human cardiac myosin heavy chain and to *T. cruzi* B13 protein and express high levels of INF- and low levels of IL-4, switching to a Th1 profile (Cunha-Neto Edecio & Kalilf, 2001).

In recent years, Treg cells have come to attention in relation to Chagas' disease pathogenesis. These cells are characterized by the expression of CD4, CD25 and FOXP3 (Ziegler & Buckner, 2009). Treg cells are increased in indeterminate patients compared to CCC, which correlates negatively with levels of activated CD8+ (Vitelli-Avelar, et al., 2005). A second subset of T CD4+ cells, recently described, the Th17 cells, resulted important in Chagas' disease pathogenesis. These cells, mainly linked to autoimmune pathology, are characterized by the expression of CD4+, RORt, and secrete IL-17 (Di Jin, et al., 2008). They were increased in a murine model of acute myocarditis induced by *T. cruzi* infection, as well as by immunization with heat-killed *T. cruzi* antigens (Bonney, et al., 2011). Both cell subsets seem to be related, as they require TGF- to differentiate. In the presence of proinflammatory cytokines, differentiation to Th17 cell prevails and a pro-autoimmune profile develops (Ziegler & Buckner, 2009).

An additional mechanism is the bystander activation. This is the activation of autoreactive lymphocytes by antigen presenting cells in a proinflammatory environment (Fujinami, et al., 2006). This kind of autoreactive T cells activation has been described in Chagas'disease (Fedoseyeva, et al., 1999).
