Human Dendritic Cell Maturation Is Modulated by Leishmania mexicana through Akt Signaling Pathway.

Dendritic cells (DC) along with macrophages are the main host cells of the intracellular parasite Leishmania. DC traverse a process of maturation, passing through an immature state with phagocytic ability to a mature one where they can modulate the immune response through the secretion of cytokines. Several studies have demonstrated that Leishmania inhibits DC maturation. Nevertheless, when cells are subjected to a second stimulus such as LPS/IFN-γ, they manage to mature. In the maturation process of DC, several signaling pathways have been implicated, importantly MAPK. On the other hand, Akt is a signaling pathway deeply involved in cell survival. Some Leishmania species have shown to activate MAPK and Akt in different cells. The aim of this work was to investigate the role of ERK and Akt in the maturation of monocyte-derived DC (moDC) infected with L. mexicana. moDC were infected with L. mexicana metacyclic promastigotes, and the phosphorylation of ERK and Akt, the expression of MHCII and CD86 and IL-12 transcript, and secretion were determined in the presence or absence of an Akt inhibitor. We showed that L. mexicana induces a sustained Akt and ERK phosphorylation, while the Akt inhibitor inhibits it. Moreover, the infection of moDC downregulates CD86 expression but not MHCII, and the Akt inhibitor reestablishes CD86 expression and 12p40 production. Thus, L. mexicana can modulate DC maturation though Akt signaling.

Involvement of Akt and the antiapoptotic protein Bcl-xL in the inhibition of apoptosis of dendritic cells by Leishmania mexicana.

The intracellular parasite Leishmania mexicana inhibits camptothecin (CPT)-induced apoptosis of monocyte-derived dendritic cells (moDC) through the down-regulation of p38 and JNK phosphorylation, while the kinase Akt is maintained active for 24 h. In addition, the infection of moDC with L. mexicana promastigotes increases the protein presence of the antiapoptotic protein Bcl-xL. In the present work, we aimed to investigate the role of Akt in the inhibition of apoptosis of moDC by L. mexicana and in the modulation of the expression of the antiapoptotic proteins Bcl-2, Mcl-1 and Bcl-xL. moDC were infected with L. mexicana metacyclic promastigotes and treated with CPT, an Akt inhibitor, or both and the mitochondrial outer membrane permeabilization (MOMP) and protein presence of active caspase 3, Bcl-2, Mcl-1 and Bcl-xL were evaluated. Our results show that the specific inhibition of Akt reverts the apoptosis protective effect exerted by L. mexicana on moDC reflected by a reduction in MOMP, caspase 3 activation, and upregulation of Bcl-xL. Interestingly, we also found that the infection of moDC with L. mexicana promastigotes induces a decrease in Bcl-2 along with an isoform change of Mcl-1, this independently to Akt activity. We demonstrated that Akt is deeply involved in the inhibition of apoptosis of moDC by L. mexicana.

Differential Regulation of l-Arginine Metabolism through Arginase 1 during Infection with Leishmania mexicana Isolates Obtained from Patients with Localized and Diffuse Cutaneous Leishmaniasis.

l-Arginine metabolism through arginase 1 (Arg-1) and inducible nitric oxide synthase (NOS2) constitutes a fundamental axis for the resolution or progression of leishmaniasis. Infection with Leishmania mexicana can cause two distinct clinical manifestations: localized cutaneous leishmaniasis (LCL) and diffuse cutaneous leishmaniasis (DCL). In this work, we analyzed in an in vivo model the capacity of two L. mexicana isolates, one obtained from a patient with LCL and the other from a patient with DCL, to regulate the metabolism of l-arginine through Arg-1 and NOS2. Susceptible BALB/c mice were infected with L. mexicana isolates from both clinical manifestations, and the evolution of the infection as well as protein presence and activity of Arg-1 and NOS2 were evaluated. The lesions of mice infected with the DCL isolate were bigger, had higher parasite loads, and showed greater protein presence and enzymatic activity of Arg-1 than the lesions of mice infected with the LCL isolate. In contrast, NOS2 protein synthesis was poorly or not induced in the lesions of mice infected with the LCL or DCL isolate. The immunochemistry analysis of the lesions allowed the identification of highly parasitized macrophages positive for Arg-1, while no staining for NOS2 was found. In addition, we observed in lesions of patients with DCL macrophages with higher parasite loads and stronger Arg-1 staining than those in lesions of patients with LCL. Our results suggest that L. mexicana isolates obtained from patients with LCL or DCL exhibit different virulence or pathogenicity degrees and differentially regulate l-arginine metabolism through Arg-1.

GK1 Improves the Immune Response Induced by Dendritic Cells of BALB/c Mice Infected with Leishmania mexicana Promastigotes.

PURPOSE: Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs), and their capacity to activate the immune response has been widely used in immunotherapies against different diseases, predominantly cancer. However, they have not been so widely used in immunotherapies against infectious diseases. Leishmania mexicana is the causative agent of cutaneous leishmaniasis in Mexico, which can result in localized cutaneous leishmaniasis (LCL) and diffuse cutaneous leishmaniasis (DCL). DCL is characterized by the incapability of the immune response to control the parasite, which thus disseminates to all teguments. Treatments against DCL have shown low efficacy, which is a reason why alternative therapies such as immunotherapies are promising. One adjuvant that has proven its effectiveness in immunotherapies against some cancers and infections is GK1, a component of the SPVac vaccine against porcine cysticercosis. GK1 has the capacity to elicit proinflammatory cytokines and chemokines from DCs and macrophages. METHODS: We pulsed bone marrow-derived dendritic cells (BMDCs) with GK1 and a lysate obtained from L. mexicana promastigotes and tested the efficacy of this combination against the infection of susceptible mice with L. mexicana. RESULTS: We found that BMDCs stimulated with GK1 and a lysate of L. mexicana promastigotes secreted IFN-γ and IL-12, and when they were adoptively transferred to BALB/c mice which were then infected with L. mexicana promastigotes, there was a reduction in the size of the lesion and in the parasite load. CONCLUSIONS: The adjuvant properties of GK1 along with parasite antigens may have a protective effect against the infection of BALB/c mice with L. mexicana.

Effect of Two Different Isolates of Leishmania mexicana in the Production of Cytokines and Phagocytosis by Murine Dendritic Cells.

Species of the genus Leishmania are the causal agents of leishmaniasis, a disease with diametrically different clinical manifestations that have been attributed to the species and host immune response. Some Leishmania species, including Leishmania mexicana, are capable of causing both localized cutaneous leishmaniasis (LCL) and diffuse cutaneous leishmaniasis (DCL). Therefore, it is possible that intraspecific differences may exist that contribute to the development of distinct clinical forms. Dendritic cells (DC) are important host cells of Leishmania spp. parasites, and cytokine production and phagocytosis upon infection with the parasite are significant for the outcome of the disease. In the present study we analyzed the production of IL-12, TNF-α, and IL-10 by DC infected with L. mexicana amastigotes isolated from a patient with LCL (amastigote = Lac) and from a patient with DCL (amastigote = Diact) by murine DC. Furthermore, we compared the frequency of phagocytosis of L. mexicana amastigotes of each isolate by fluorescence and optical microscopy and by flow cytometry. We show that the infection of DC with Diact amastigotes elicited the secretion of IL-10, TNF-α, and IL-12 by DC to a major extent as compared to the infection with Lac amastigotes. On the other hand, Lac and Diact amastigotes were similarly phagocytosed by DC, but interestingly there were more vacuoles in DC infected with Diact amastigotes. Our results suggest that isolates from a same species of Leishmania, such as L. mexicana, with different degrees of virulence according to the clinical manifestation they cause, differ in their capacity to elicit cytokine production and form vacuoles in DC.

Role of glutathione, ROS, and Bcl-xL in the inhibition of apoptosis of monocyte-derived dendritic cells by Leishmania mexicana promastigotes.

Dendritic cells (DCs) are one of the principal host cells of the obligate intracellular parasite Leishmania that can survive and reproduce within cells due to the ability to regulate different cellular events, including apoptosis. Inhibition of host cell apoptosis is a strategy employed by multiple pathogens to ensure their survival in the infected cell. We have previously reported that Leishmania mexicana promastigotes and amastigotes inhibit camptothecin-induced apoptosis of monocyte-derived dendritic cells (moDCs) through the downregulation of p38 and JNK phosphorylation. The upregulation of glutathione (GSH), the most important regulator of reactive oxygen species (ROS) concentration, has proven to protect cells from apoptosis through the inhibition of JNK1. Another mechanism employed by cells for the protection of apoptosis is the expression of anti-apoptotic proteins of the Bcl-2 family. The aim of this study was to determine if GSH, ROS, and Bcl-xL participate in the inhibition of camptothecin-induced apoptosis of moDC by L. mexicana promastigotes. GSH quantification assays showed that camptothecin and BSO (an inhibitor of glutathione synthesis) strongly decreased intracellular GSH concentration in moDC, while infection with L. mexicana promastigotes had no effect in the level of GSH. On the other hand, infection with L. mexicana promastigotes of BSO- and camptothecin-treated moDC diminished the concentration of ROS and induced the expression of the anti-apoptotic protein Bcl-xL. Our findings suggest that inhibition of camptothecin-induced apoptosis of moDC by L. mexicana promastigotes is preferentially regulated by the expression of anti-apoptotic proteins of the Bcl-2 family rather than by the redox status of the cell.

NKT cell activation by Leishmania mexicana LPG: Description of a novel pathway.

NKT cells have been associated with protection against Leishmania donovani, yet their role in infections with Leishmania mexicana has not been addressed, nor has the activation pathway been defined after stimulation with Leishmania mexicana lipophosphoglycan (LPG). We analyzed the activation of NKT cells and their cytokine production in response to Leishmania mexicana LPG. Additionally we compared NKT-cell numbers and cytokine profile in lymph nodes of skin lesions induced by Leishmania mexicana in BALB/c and C57BL/6 mice. We show that LPG activates NKT cells primarily through the indirect pathway, initiating with TLR2 stimulation of dendritic cells (DC), thereby enhancing TLR2, MHC II, and CD86 expressions and IL-12p70 production. This leads to IFN-γ production by NKT cells. C57BL/6 mice showed enhanced DC activation, which correlated with augmented IFN-γ production by NKT cells. Additionally, infected C57BL/6 mice showed elevated percentages of NKT cells with higher IFN-γ and IL-4 production in lymph nodes. We conclude that the response of NKT cells towards Leishmania mexicana LPG initiates with the indirect activation, after binding of LPG to TLR2 in DC. This indirect activation pathway enables NKT cells to produce IFN-γ during the innate phase of Leishmania infection, the magnitude of which differs between mouse strains.

Leishmania mexicana promastigotes down regulate JNK and p-38 MAPK activation: Role in the inhibition of camptothecin-induced apoptosis of monocyte-derived dendritic cells.

Dendritic cells (DC) are one of the principal host cells of the obligate intracellular parasite Leishmania. Inhibition of host cell apoptosis is a strategy employed by multiple pathogens to ensure their survival in the infected cell. We have previously shown that the infection of monocyte-derived dendritic cells (moDC) with Leishmania mexicana inhibits campthotecin-induced apoptosis. Nevertheless, the mechanisms involved in the inhibition of apoptosis of dendritic cells by Leishmania have not been established. Mitogen-activated protein kinases (MAPK) are key participants in the process of apoptosis and different species of Leishmania have been shown to regulate these kinases. In the present study, we analyzed the effect of L. mexicana promastigotes in the activation of JNK and p38 MAP kinase and their participation in the inhibition of apoptosis. The infection of moDC with L. mexicana promastigotes diminished significantly the phosphorylation of the MAP kinases JNK and p38. The inhibition of both kinases diminished DNA fragmentation, but in a major extent was the reduction of DNA fragmentation when JNK was inhibited. The capacity of L. mexicana promastigotes to diminish MAP kinases activation is probably one of the strategies employed to delay apoptosis induction in the infected moDC and may have implications for Leishmania pathogenesis by favoring the invasion of its host and the persistence of the parasite in the infected cells.

Differential phagocytosis of Leishmania mexicana promastigotes and amastigotes by monocyte-derived dendritic cells.

Leishmania species are dimorphic protozoan parasites that live and replicate in the gut of sand flies as promastigotes or in mammalian hosts as amastigotes. Different immune cells, including DCs, and receptors differ in their involvement in phagocytosis of promastigotes and amastigotes and in recognition of different Leishmania species. In the case of L. mexicana, differences in phagocytosis of promastigotes and amastigotes by DCs and participation of C-type lectin receptors (CLRs) have not been established. In the present study, flow cytometry and confocal microscopy were used to investigate the phagocytosis by monocyte-derived dendritic cells (moDCs) of L. mexicana promastigotes and amastigotes in the presence or absence of immune serum during various periods of time. Blocking antibodies against mannose receptors and DC-SIGN were used to explore the participation of these receptors in the phagocytosis of L. mexicana by moDC. The major differences in interactions of L. mexicana promastigotes and amastigotes with moDC were found to occur within the first 3 hr, during which phagocytosis of promastigotes predominated as compared with opsonization of promastigotes and amastigotes. However, after 6 hr of incubation, opsonized promastigotes were preferentially phagocytosed as compared with unopsonized promastigotes and amastigotes and after 24 hr of incubation there were no differences in the phagocytosis of promastigotes and amastigotes. Finally, after 3 hr incubation, DC-SIGN was involved in the phagocytosis of promastigotes, but not of amastigotes.

Regulation of the expression of nitric oxide synthase by Leishmania mexicana amastigotes in murine dendritic cells.

In mammalian hosts, Leishmania parasites are obligatory intracellular organisms that invade macrophages (M phi) and dendritic cells (DC). In M phi, the production of nitric oxide (NO) catalyzed by the inducible nitric oxide synthase (iNOS) has been implicated as a major defense against Leishmania infection. The modulation of this microbicidal mechanism by different species of Leishmania has been well studied in M phi. Although DC are permissive for infection with Leishmania both in vivo and in vitro, the effect of this parasite in the expression of iNOS and NO production in these cells has not been established. To address this issue, we analyzed the regulation of iNOS by Leishmania mexicana amastigotes in murine bone marrow-derived dendritic cells (BMDC) stimulated with LPS and IFN-gamma. We show that the infection of BMDC with amastigotes down regulated NO production and diminished iNOS protein levels in cells stimulated with LPS alone or in combination with IFN-gamma. The reduction in iNOS protein levels and NO production did not correlate with a decrease in iNOS mRNA expression, suggesting that the parasite affects post-transcriptional events of NO synthesis. Although amastigotes were able to reduce NO production in BMDC, the interference with this cytotoxic mechanism was not sufficient to permit the survival of L. mexicana. At 48 h post-infection, BMDC stimulated with LPS+IFN-gamma were able to eliminate the parasites. These results are the first to identify the regulation of iNOS by L. mexicana amastigotes in DC.