*7.2.1. Apoptosis induction*

in BMM and RAW 264.7 cells increased the expression of PD-1, while the infection with *L. donovani* diminished it along with the induction of apoptosis. The activation of PD-1 pathway was found to negatively regulate the phosphorylation of pro-survival AKT, which was reversed

During the induction of apoptosis, reactive oxygen species (ROS) are produced; on the other hand, an overproduction of (ROS) induces apoptosis. The analysis of the effect of *Leishmania* in

with *L. donovani* promastigotes and was observed that the parasite did not affect the level of ROS and apoptosis was inhibited along with a decrease in caspase 3 and 7, which could be due to Suppressor Of Cytokine Signaling (SOCS) along with the activity of thioredoxin and tyrosine phosphatases. The silencing of SOCS genes diminished thioredoxin levels and increased apoptosis of cells [123]. Recently, De Souza-Vieira et al. demonstrated the activation of two PI3K isoforms, PI3K γ (ROS dependent) and PI3K δ (ROS independent) in neutrophils infected with *L. amazonensis*. The activation of these isoforms, in turn, activates the ERK pathway downstream, which is associated with the process of netosis with the subsequent activation of ROS and the

Chagas' disease affects nearly 8 million people in Latin America [125] and is caused by the intracellular parasite *Trypanosoma cruzi*. The infection with *T. cruzi* is characterized by an acute phase that can be controlled by the immune system of the host. Afterwards, patients can remain asymptomatic or develop a chronic phase that affects mainly the heart and peripheral nervous system [126, 127]. In some cases, patients seem asymptomatic although they present several damages. *T. cruzi* has the capacity to infect virtually any cell where infective tripomastigotes reach the cytoplasm, replicate, lyse the cell and infect other cells. One of the cells inside the mammalian host where *T. cruzi* replicates is the macrophage. These cells 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 capacity of killing *T. cruzi*, whereas alternatively activated macrophages, belonging to the M2 spectrum, synthesize polyamines that actually promote infection [128, 129]. Thus, one of the most important mechanisms of protective immunity against *T. cruzi* is the classical activation of macrophages for the elimination of the intracellular parasites. *T. cruzi* must control the activation of macrophages and inhibit apoptosis in order to perpetuate inside the cells. To achieve this, parasites must reduce the production of toxic molecules, including NO and its derivatives [130, 131] and must escape from the parasitophorous vacuole [132].

The development of a specific immune response against *T. cruzi* overcomes the evasion strategies displayed by the parasite. Antibodies as well as T cells are required for the control of infection [133–135], where both CD4 and CD8 T cells produce IFN-*γ* that activates macrophages to restrain infection [136, 137], while CD8 T cells eliminate cells harboring parasites in the cytoplasm and also promote immunopathology in the heart [138, 139]. Some of these

O2

and infected

the modulation of ROS was analyzed in RAW 264.7 macrophages treated with H2

release of neutrophil extracellular traps (NETs) [124].

**7.1. Immune response to** *T. cruzi*

**7. Inhibition of apoptosis by** *Trypanosoma cruzi*

during infection [122].

104 Current Understanding of Apoptosis - Programmed Cell Death

As just mentioned, it has been demonstrated that there is intense apoptosis of T lymphocytes during the course of *T. cruzi* infection [142]. The induction of apoptosis occurs through the extrinsic pathway as the infection with *T. cruzi* provokes the expression of both Fas (CD95) and Fas ligand (FasL) [143, 145], caspase-8 activity, and activation of effector caspase-3 [143, 144] in T lymphocytes from *T. cruzi*-infected mice. CD8 T lymphocytes help in the control of infection by *T. cruzi*, and the induction of apoptosis of these cells disrupts the immune response and interestingly affects macrophage activation. Apoptosis of CD8 T lymphocytes promotes macrophage differentiation toward an M2-like phenotype, which favors *T. cruzi* infection [150].

#### *7.2.2. Apoptosis inhibition*

*T. cruzi* resides in the cytoplasm of diverse cells, and thus CD8 lymphocytes are important for their elimination. It has been shown that CD8 T cells are preferential targets, as compared to CD4 lymphocytes, for early effects of apoptosis inhibition in acute infection [146, 151]. In addition to macrophages, *T. cruzi* infects cardiomyocytes and it is common to find an intense myocarditis during the acute phase of infection. Despite cardiac damage, infected individuals may remain asymptomatic for decades. Thus, *T. cruzi* may directly prevent cardiomyocyte death in order to prevent heart destruction and favor its survival. It has been shown that *T. cruzi,* as well as cruzipain, an important *T. cruzi* antigen, promotes survival of cardiomyocytes cultured under serum deprivation through the expression of the antiapoptotic protein Bcl-2, but not of Bcl-xL. Also, *T. cruzi* displays other antiapoptotic strategies such as the phosphorylation of Akt and ERK 1/2, which differentially modulate Bcl-2 family members [152]. In addition, cruzipain enhances arginase activity that favors parasite growth within the cell. Interestingly, the inhibition of arginase activity by NG-hydroxy-L-arginine (NOHA) abrogated the antiapoptotic action of cruzipain suggesting that arginase activity is required for the survival effect of cruzipain [153].

Apart from invading the heart, *T. cruzi* colonizes the peripheral nervous system and it has been shown that the infection of Schwann cells by *T. cruzi* suppresses host cell apoptosis caused by growth factor deprivation. The antiapoptotic effect of the parasite has been related to the interaction of Akt with *T. cruzi* PDNF, glycosylphosphatidylinositol (GPI)-anchored parasite-derived neurotrophic factor, known mostly for its neuraminidase and sialyltransferase activities [154].
