Magdalena Aguirre-García

Leishmania mexicana: Inhibition of camptothecin-induced apoptosis of monocyte-derived dendritic cells

Macrophages (Mphi) and dendritic cells (DC) are the major target cell populations of the obligate intracellular parasite Leishmania. Inhibition of host cell apoptosis is a method employed by multiple pathogens to ensure their survival in the infected cell. Leishmania has been shown to protect Mphi and neutrophils from both natural and induced apoptosis. As shown in this study, apoptosis in monocyte-derived dendritic cells (moDC) induced by treatment with camptothecin was downregulated by coincubation with L. mexicana, as detected by morphological analysis of cell nuclei, TUNEL assay, gel electrophoresis of low molecular weight DNA fragments, and annexin V binding to phosphatidylserine. The observed antiapoptotic effect was found to be associated with a significant reduction of caspase-3 activity in moDC. The capacity of L. mexicana to delay apoptosis induction in the infected moDC may have implications for Leishmania pathogenesis by favoring the invasion of its host and the persistence of the parasite in the infected cells.

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.

Leishmania mexicana: Participation of NF-κB in the differential production of IL-12 in dendritic cells and monocytes induced by lipophosphoglycan (LPG)

Dendritic cells (DC) and macrophages (Mphi) are well known as important effectors of the innate immune system and their ability to produce IL-12 indicates that they possess the potential of directing acquired immunity toward a Th1-biased response. Interestingly, the intracellular parasite Leishmania has been shown to selectively suppress Mphi IL-12 production and are DC the principal source of this cytokine. The molecular details of this phenomenon remain enigmatic. In the present study we examined the effect of Leishmania mexicana lipophosphoglycan (LPG) on the production of IL-12, TNF-alpha, and IL-10 and nuclear translocation of NF-kappaB. The results show that LPG induced more IL-12 in human DC than in monocytes. This difference was due in part to nuclear translocation of NF-kappaB, since LPG induced more translocation in DC than in monocytes. These results suggest that Leishmania LPG impairs nuclear translocation of NF-kappaB in monocytes with the subsequent decrease in IL-12 production.

Leishmania major : detection of membrane-bound protein tyrosine phosphatase.

PTPases have been reported as a virulence factor in different pathogens. Recent studies suggest that PTPases play a role in the pathogenesis of Leishmania infections through activation of macrophage PTPases by the parasite. We report here the presence of a membrane-bound PTPase in Leishmania major promastigotes. We detected differences in the PTPases present in the procyclic and metacyclic stages of promastigotes. In metacyclic promastigotes, the PTPase activity was totally inhibited by specific PTPase and serine/threonine inhibitors, whereas in procyclic promastigotes the PTPase activity was inhibited only with PTPase inhibitors. Two antibodies against the catalytic domains of the human placental PTPase1B and a PTPase from Trypanosoma brucei cross-reacted with a 55-60 kDa molecule present in the soluble detergent-extracted fraction of a Leishmania homogenate. Metacyclic promastigotes expressed more of this molecule than parasites in the procyclic stage. Yet the specific activity of the enzyme was lower in metacyclic than in procyclic promastigotes. Ultrastructural localization of the enzyme showed that it was more membrane-associated in metacyclic promastigotes, whereas in procyclic promastigotes it was scattered throughout the cytoplasm. This is the first demonstration of a PTPase present in Leishmania major promastigotes that differs in expression, activity and ultrastructural localization between the procyclic and metacyclic stages of the parasites life-cycle.

Leishmania mexicana lipophosphoglycan activates ERK and p38 MAP kinase and induces production of proinflammatory cytokines in human macrophages through TLR2 and TLR4

Protozoan parasites of genus Leishmania are the causative agents of leishmaniasis. Leishmania promastigotes primarily infect macrophages in the host, where they transform into amastigotes and multiply. Lipophosphoglycan (LPG), the most abundant surface molecule of the parasite, is a virulence determinant that regulates the host immune response. Promastigotes are able to modulate this effect through LPG, creating a favourable environment for parasite survival, although the mechanisms underlying this modulation remain unknown. We analysed the participation of TLR2 and TLR4 in the production of cytokines and explored the possible phosphorylation of ERK and/or p38 MAP kinase signalling cascades in human macrophages stimulated with Leishmania mexicana LPG. The results show that LPG induced the production of TNF-α, IL-1β, IL-12p40, IL-12p70 and IL-10 and led to phosphorylation of ERK and p38 MAP kinase. Specific inhibitors of ERK or p38 MAP kinases and mAbs against TLR2 and TLR4 reduced cytokine production and phosphorylation of both kinases. Our results suggest that L. mexicana LPG binds TLR2 and TLR4 receptors in human macrophages, leading to ERK and MAP kinase phosphorylation and production of pro-inflammatory cytokines.

Leishmania mexicana amastigotes inhibit p38 and JNK and activate PI3K/AKT: role in the inhibition of apoptosis of dendritic cells

Leishmania mexicana is the causal agent of cutaneous leishmaniasis in Mexico. Dendritic cells (DC) are one of the host cells of Leishmania parasites. Intracellular microorganisms inhibit host cell apoptosis as a strategy to ensure their survival in infected cells. We have previously shown that Leishmania mexicana promastigotes and amastigotes inhibit camptothecin‐induced apoptosis of monocyte‐derived dendritic cells (moDC), but the mechanisms underlying the inhibition of apoptosis of DC by Leishmania have not been established. MAP kinases and PI3K participate in the process of apoptosis and are modulated by different species of Leishmania. As shown in this study, the infection of moDC with L. mexicana amastigotes diminished significantly the phosphorylation of the MAP kinases p38 and JNK. The inhibition of both kinases diminished significantly DNA fragmentation in moDC stimulated with camptothecin. On the other hand, L. mexicana amastigotes were able to activate the anti‐apoptotic pathways PI3K and AKT. Our results indicate that L. mexicana amastigotes have the capacity to diminish MAP kinases activation and activate PI3K and AKT, which is probably one of the strategies employed by L. mexicana amastigotes to inhibit apoptosis in the infected moDC.

NK Cell Activity Differs between Patients with Localized and Diffuse Cutaneous Leishmaniasis Infected with Leishmania mexicana: A Comparative Study of TLRs and Cytokines

Leishmania mexicana causes localized (LCL) or diffuse cutaneous leishmaniasis (DCL). The cause of dissemination in DCL remains unknown, yet NK cells possibly play a role in activating leishmanicidal mechanisms during innate and adaptive immune responses. We had previously shown that Leishmania lipophosphoglycan (LPG) is a ligand for TLR2, activating human NK cells. We have now analyzed NK cells in LCL and DCL patients. NK numbers and effector mechanisms differed drastically between both groups of patients: DCL patients showed reduced NK cell numbers; diminished IFN-γ and TNF-α production; and lower TLR2, TLR1, and TLR6 expression as compared to LCL patients. The altered protein expression found in NK cells of DCL patients correlated with their down-regulation of IFN-γ gene expression in LPG-stimulated and non-stimulated cells as compared to LCL patients. NK cell response was further analyzed according to gender, age, and disease evolution in LCL patients showing that female patients produced higher IFN-γ levels throughout the disease progression, whereas TLR2 expression diminished in both genders with prolonged disease evolution and age. We furthermore show the activation pathway of LPG binding to TLR2 and demonstrated that TLR2 forms immunocomplexes with TLR1 and TLR6. In addition to the reduced NK cell numbers in peripheral blood, DCL patients also showed reduced NK cell numbers in the lesions. They were randomly scattered within the lesions, showing diminished cytokine production, which contrasts with those of LCL lesions, where NK cells produced IFN-γ and TNF-α and were found within organized granulomas. We conclude that in DCL patients the reduced NK-cell numbers and their diminished activity, evidenced by low TLR expression and low cytokine production, are possibly involved in the severity of the disease. Our results provide new information on the contribution of NK cells in Leishmania infections of the human host.

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.

Leishmania mexicana: promastigotes and amastigotes secrete protein phosphatases and this correlates with the production of inflammatory cytokines in macrophages.

Phosphatase activity of Leishmania spp. has been shown to deregulate the signalling pathways of the host cell. We here show that Leishmania mexicana promastigotes and amastigotes secrete proteins with phosphatase activity to the culture medium, which was higher in the Promastigote Secretion Medium (PSM) as compared with the Amastigote Secretion Medium (ASM) and was not due to cell lysis, since parasite viability was not affected by the secretion process. The biochemical characterization showed that the phosphatase activity present in PSM was higher in dephosphorylating the peptide END (pY) INASL as compared with the peptide RRA (pT)VA. In contrast, the phosphatase activity in ASM showed little dephosphorylating capacity for both peptides. Inhibition assays demonstrated that the phosphatase activity of both PSM and ASM was sensible only to protein tyrosine phosphatases inhibitors. An antibody against a protein phosphatase 2C (PP2C) of Leishmania major cross-reacted with a 44·9 kDa molecule in different cellular fractions of L. mexicana promastigotes and amastigotes, however, in PSM and ASM, the antibody recognized a protein about 70 kDa. By electron microscopy, the PP2C was localized in the flagellar pocket of amastigotes. PSM and ASM induced the production of tumor necrosis factor alpha, IL-1β, IL-12p70 and IL-10 in human macrophages.

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.