**5.1. Inhibition of apoptosis by** *Leishmania*

*Leishmania* is an obligate intracellular parasite that infects a variety of cells such as neutrophils, macrophages (Mφ) and dendritic cells (DC). *Leishmania* has developed diverse mechanisms to manipulate host cells in order to evade the immune response and survive inside cells. Some of these strategies are the evasion of the phagosome-lysosome fusion and the inhibition of apoptosis. Studies have demonstrated that monocytes, macrophages, and dendritic cells grown in apoptogenic conditions and infected with different species of *Leishmania* present an inhibition of normal apoptosis. Also, *Leishmania* infection prevents natural apoptosis of neutrophils. The first demonstration of the inhibition of apoptosis by *Leishmania* was performed by Moore and Matlashewski in 1994 who demonstrated that the infection of bone marrow derived-macrophages (BMM) with *Leishmania donovani* promastigotes or the stimulus with LPG inhibited apoptosis induced by the deprivation of M-CSF. Interestingly, the culture supernatant of infected BMM was able to inhibit apoptosis suggesting that the effect could be due to soluble mediators [99]. A later study showed that cellular activation increased the production of TNF-α, TGF-β, IL-6, and GM-CSF, while the secretion of M-CSF and IL-1β diminished [100]. Studies performed later with another species, *Leishmania major,* showed that the infection of macrophages grown in the absence of M-CSF or in the presence of staurosporine inhibited the release of mitochondrial cytochrome c, thus delaying apoptosis. It was observed that the infection of BMM with *L. major* promastigotes inhibited caspase-3 activation owed to a decrease in MOMP and subsequent release of cytochrome c, which was not associated to NF-κB activation since the use of specific inhibitors did not affect the capacity of L. major to inhibit macrophage apoptosis. It was also demonstrated that the infection of BMM obtained from BALB/c o C57BL/6 mice with *L. major* promastigotes preserved the phenomenom of apoptosis inhibition despite the genetic background of the host or type of immune response (Th2 or Th1, respectively) [101]. Also, studies performed with cell lines reported 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 apoptosis as compared to the infection with the more virulent (LV39) [103].

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 pro-

Signaling Pathways Targeted by Protozoan Parasites to Inhibit Apoptosis

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apoptotic and also inhibiting the production of IL-12 [119].

serum were very low [121].

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

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
