**6. Signaling pathways involved in the inhibition of apoptosis by**  *Leishmania*

As it has been just mentioned, *Leishmania* has the capacity to inhibit apoptosis of different cells; however, the mechanisms involved in this inhibition have not been fully deciphered. MAPK and PI3K have been implicated due to their participation in apoptosis and the intervention of *Leishmania* with these kinases. Regarding the role of *Leishmania* infection in the modulation of proapoptotic pathways such as MAPK, it has been shown that *L. mexicana* amastigotes and promastigotes significantly reduced MAPK JNK and p38 phosphorylation in monocytederived dendritic cells [108, 109]. Other authors working with the same species showed that the inhibitoy effect in the activation of MAPK in dendritic cells was only observable in immature dendritic cells since maturation driven by the stimulation with LPS did not suppress MAPK phosphorylation, in particular JNK [110]. In bone marrow macrophages (BMM), previously stimulated with IFN-γ, it was also shown that *L. donovani* promastigotes exerted a similar effect of inhibiting the activation of p38, JNK, and ERK that was directly associated with TNF-α production, which ensured the survival of the parasite [111]. Other authors also demonstrated that inhibition of p38 was associated with an increase in the number of infected macrophages and parasite survival [112]. Interestingly, not only the parasite but also some surface components such as gp63 have been shown to inhibit the apoptotic signaling of MAPK p38 [113]. Other studies have shown that *Leishmania* infection can also activate MAPK as demonstrated with the infection of neutrophils with *L. major* that caused the transient activation of ERK1/2, which delayed apoptosis and the pharmacological inhibition of ERK1/2 phosphorylation reversed the effect. Moreover, the infection of neutrophils with *L. major* led to the enhanced and sustainable expression of the antiapoptotic proteins Bcl-2 and Bfl-1. As downstream events, the release of cytochrome c from mitochondria and processing of caspase-6 were inhibited, as well as a reduced expression of FAS on the surface of neutrophils [114]. In BMM the infection with infected with *L. amazonensis* it has been observed that ERK 1/2 activation generates an epigenetic modification in the IL-10 locus, which results in a great induction of this cytokine in the infected macrophages [115]. Also, macrophages grown in the presence of LPG show an altered production of IL-12 associated with ERK activation and signaling [116]. Other authors demonstrated that ERK 1/2 activation induced by *L. amazonensis* yielded a lesser expression of CD40 and IL-12 production in bone marrow derived dendritic cells, with the subsequent inhibition of dendritic cell maturation. Specific ERK 1/2 inhibition induced the production of NO which caused an increase in parasite death [117]. Interestingly, *Leishmania* infection not only intervenes with signaling pathways that induce apoptosis but also with pathways that promote survival as it has been shown with the infection of BMM with *L. major* and *L. pifanoi* promastigotes that promotes resistance to apoptosis through activation of PI3K/ Akt. It was also demonstrated that Akt phosphorylates Bad, which in turn interacts with the 14–3-3 protein, inhibiting it and boosting the antiapoptotic action of Bcl-2 [118]. It has also been demonstrated that infection of monocyte-derived dendritic cells with *L. mexicana* amastigotes activated antiapoptotic signals, such as PI3K/Akt phosphorylation [108]. Recently, the participation of Akt in the inhibition of apoptosis by *Leishmania* has been more widely analyzed. The infection of BMM or RAW 264.7 with *L. donovani* promastigotes and treated both with a specific Akt inhibitor or a dominant negative construct diminished the antiapoptotic effect, increased the production of IL-12, and decreased the production of IL-10, which resulted in loss of parasite survival. It was shown that in infected cells FOXO-1, a transcriptional regulator of proapoptotic proteins, is found mainly in the cytoplasm. The transfection of cells with FOXO-1, constitutively active that cannot be phosphorylated Akt and thus remained sequestered in the nucleus, led to a reduction of the antiapoptotic effect in infected period. Also, it was observed that the activation of Akt, induced by the infection of macrophages with L. *donovani* promastigotes, causes the inactivation of GSK-3β (Glycogen synthase kinase 3 beta), which permits the release of β-catenin in order to initiate the transcription of antiapoptotic proteins. It was shown that in infected cells and transfected with the constituvely active construct for GSK-3β by silencing β-catenin there was a loss in mitochondrial membrane potential along with the activation of caspase-3 and production of IL-12 [119]. This was the first observation showing that the reversion of the antiapoptotic effect diminishes parasite survival, which suggests that the Akt pathway is a pivotal step in the modulation of the cellular machinery since *Leishmania* through the modulation of Akt is capable of activating antiapoptotic proteins, inhibiting proapoptotic and also inhibiting the production of IL-12 [119].

similar results as in the case of the monocyte cell line U937 infected with *Leishmania infantum* where inhibition of actinomycin D-induced apoptosis was observed [102] or in macrophages from the cell line RAW 264.7 infected with *Leishmania major* where apoptosis diminished even in the presence of cycloheximide [103]. In neutrophils, it has been observed that spontaneous apoptosis is inhibited by *Leishmania major* due to a decrease in caspase-3 activity [104]. It has also been demonstrated that amastigotes and promastigotes of *Leishmania mexicana* inhibit camptothecin-induced apoptosis in monocyte-derived dendritic cells [105, 106]. Moreover, *Leishmania* parasites are characterized for presenting differences among different species and also intraspecific. In particular, it has been shown that different strains of *L. major* cause diverse clinical manifestations in susceptible BALB/c mice [107]. While the infection with the strains V1 and IR137 could be resolved, the infection with the LV39 strain presents a severe course of infection, which cannot be resolved. The infection of RAW 264.7 macrophages with the less virulent strains (V1 e IR37) of *L. major* showed a lower degree of inhibition of apopto-

sis as compared to the infection with the more virulent (LV39) [103].

102 Current Understanding of Apoptosis - Programmed Cell Death

*Leishmania*

**6. Signaling pathways involved in the inhibition of apoptosis by** 

As it has been just mentioned, *Leishmania* has the capacity to inhibit apoptosis of different cells; however, the mechanisms involved in this inhibition have not been fully deciphered. MAPK and PI3K have been implicated due to their participation in apoptosis and the intervention of *Leishmania* with these kinases. Regarding the role of *Leishmania* infection in the modulation of proapoptotic pathways such as MAPK, it has been shown that *L. mexicana* amastigotes and promastigotes significantly reduced MAPK JNK and p38 phosphorylation in monocytederived dendritic cells [108, 109]. Other authors working with the same species showed that the inhibitoy effect in the activation of MAPK in dendritic cells was only observable in immature dendritic cells since maturation driven by the stimulation with LPS did not suppress MAPK phosphorylation, in particular JNK [110]. In bone marrow macrophages (BMM), previously stimulated with IFN-γ, it was also shown that *L. donovani* promastigotes exerted a similar effect of inhibiting the activation of p38, JNK, and ERK that was directly associated with TNF-α production, which ensured the survival of the parasite [111]. Other authors also demonstrated that inhibition of p38 was associated with an increase in the number of infected macrophages and parasite survival [112]. Interestingly, not only the parasite but also some surface components such as gp63 have been shown to inhibit the apoptotic signaling of MAPK p38 [113]. Other studies have shown that *Leishmania* infection can also activate MAPK as demonstrated with the infection of neutrophils with *L. major* that caused the transient activation of ERK1/2, which delayed apoptosis and the pharmacological inhibition of ERK1/2 phosphorylation reversed the effect. Moreover, the infection of neutrophils with *L. major* led to the enhanced and sustainable expression of the antiapoptotic proteins Bcl-2 and Bfl-1. As downstream events, the release of cytochrome c from mitochondria and processing of caspase-6 were inhibited, as well as a reduced expression of FAS on the surface of neutrophils [114]. In BMM the infection with infected with *L. amazonensis* it has been observed that ERK 1/2 activation generates an epigenetic modification in the IL-10 locus, which results in a great induction of this cytokine in the infected macrophages [115]. Also, macrophages grown in the presence

Continuing with the role of *Leishmania* in modulating antiapoptotic pathways it has been shown that in murine macrophages infected with *L. donovani* promastigotes and treated with the apoptosis inductor, actinomycin D, there was an increase in the mRNA levels and protein level of MCL-1. Interestingly, the silencing of MCL-1 in infected cells dampened the antiapoptotic effect in a similar way as the silencing of the transcription factor CREB, which diminished the expression of MCL-1 and increased the apoptosis of cells [120]. In a different study, several populations of macrophages (derived from peripheral blood, THP-1 and murine) were infected with *L. donovani* and the expression of Bcl-2 increased twice with respect to uninfected cells. Also, the use of specific inhibitors for Bcl-2 increased the level of NO, which diminished the parasite load of the cells. Interestingly, it was shown that in patients with visceral leishmaniasis, there was an increase in the expression of Bcl-2 and the levels of NO in serum were very low [121].

Recently, the receptor of programmed death 1 (PD-1) has been associated with the effect of inhibition of apoptosis in cells infected with *Leishmania.* The induction of apoptosis with H2 O2

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 infection [122].

mechanisms are regulated by cytokines, such as IL-10 and TGF-β, which diminish inflammation and thus pathology, but might contribute to the persistence of parasites [140, 141]. Also, it has been demonstrated that apoptosis of lymphocytes in the course of *T. cruzi* infection down regulates T-cell expansion [142, 143], B-cell response [144], parasite killing by M1 [143, 145], and CD8 T-cell-mediated immunity [146, 147]. Furthermore, infection is also promoted by the phagocytosis by macrophages of apoptotic T lymphocytes in a matter dependent of prostaglandins, TGF-*β*, and polyamine biosynthesis [148], which are characteristic of M2 activation [128]. In contrast, the blockade of prostaglandin production or the inhibition of T lymphocyte apoptosis by caspase inhibitors reduces parasite growth *in vitro* and parasitemia in an experi-

Signaling Pathways Targeted by Protozoan Parasites to Inhibit Apoptosis

http://dx.doi.org/10.5772/intechopen.76649

105

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].

*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].

mental model of Chagas disease [148, 149].

*7.2.1. Apoptosis induction*

*7.2.2. Apoptosis inhibition*

**7.2. Apoptosis modulation in** *T. cruzi* **infection**

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 the modulation of ROS was analyzed in RAW 264.7 macrophages treated with H2 O2 and infected 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 release of neutrophil extracellular traps (NETs) [124].
