**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<sup>+</sup> and CD8<sup>+</sup> 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 transsialidase (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].

*Modulation of Host Cell Apoptosis by* Trypanosoma cruzi*: Repercussions in the Development… DOI: http://dx.doi.org/10.5772/intechopen.103740*

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 inducedapoptosis (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<sup>+</sup> 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].
