Laila Gutiérrez-Kobeh

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.

Mast cells are activated by Leishmania mexicana LPG and regulate the disease outcome depending on the genetic background of the host

The regulatory effect of mast cells on the pathogenesis of leishmaniasis is unclear. We report a comparative analysis of TLR2 membrane expression, TNF‐α, IL‐10 and MIP‐1α production, and granule release of bone marrow‐derived mast cells (BMMCs) from susceptible BALB/c and resistant C57BL/6 mice, stimulated in vitro with Leishmania mexicana lipophosphoglycan (LPG). We studied the kinetics of mast cell degranulation and parasite numbers in lesions of both mouse strains infected with L. mexicana. We found that BMMCs of C57BL/6 mice expressed more TLR2 and produced higher levels of both cytokines and MIP‐1α, whereas BALB/c BMMCs significantly augmented their granule release. Lesions of BALB/c mice showed higher levels of degranulated mast cells at 3 h of infection, whereas after 3 days of infection, the number of degranulated mast cells in C57BL/6 was higher than in BALB/c lesions. Throughout infection, BALB/c mice harboured more parasites. The regulatory effect of mast cells seems to depend on the genetic background of the host: mast cells of BALB/c mice facilitate disease progression due to an augmented inflammatory response early in the infection, whereas mast cells of C57BL/6 mice produce cytokines that regulate inflammation and maintain an elevated number of immune cells in the lesions, promoting disease control.

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.

Role of brain-derived neurotrophic factor in the protective action of N-methyl-D-aspartate in the apoptotic death of cerebellar granule neurons induced by low potassium.

Several neurotrophic factors, including brain‐derived neurotrophic factor (BDNF), and neurotransmitters, such as glutamate, may influence neuronal apoptotic death. Rat cerebellar granule neurons (CGN) cultured in low potassium (5 or 10 mM KCl) for more than 5 days in vitro (DIV) die apoptotically. These cells survive in the presence of high potassium (25 mM KCl, K25) or N‐methyl‐D‐aspartate (NMDA), an agonist of glutamatergic receptors. CGN transferred from high to low potassium die apoptotically. Here, we characterized the effect of BDNF and NMDA on the apoptotic death induced by low potassium in CGN. Cell death of CGN by culturing in low potassium for 6 DIV was inhibited by BDNF and NMDA. When CGN were cultured in K25 and transferred to a low‐potassium medium, 65% of neurons died after 48 hr. Under these conditions, BDNF, NMDA, or BDNF + NMDA increased CGN survival. Both BDNF and NMDA decreased caspase‐9 activity and mRNA caspase‐3 levels and activity induced by low potassium. CGN survival induced by BDNF is mediated by TrkB activation, whereas that induced by NMDA is mediated by NMDA receptor and TrkB activation. NMDA, but not BDNF, raised [Ca2+]i, which was reduced by low‐potassium treatment. These results suggest that NMDA receptor stimulation induces CGN survival through the influx of extracellular Ca2+ that may evoke the release of BDNF and the activation of TrkB. Complementary mechanisms induced by depolarization and changes in Ca2+ levels would also contribute to the neuroprotection exerted by NMDA and potassium.

Characterization of the activation of glutaminase induced by N-methyl-D-aspartate and potassium in cerebellar granule cells

Chronic stimulation of cerebellar granule cells with N‐methyl‐D‐aspartate (NMDA) or KCl induces a specific activation of the enzymes directly involved in glutamate neurotransmitter synthesis. Phosphate‐activated glutaminase (PAG) activity is enhanced in cultured granule neurons incubated with 150 μM NMDA or 25 mM KCl. Other enzymes are not affected by this treatment like lactate dehydrogenase (LDH) and glutamate dehydrogenase (GLDH), which is also a mitochondrial enzyme but not directly involved in neurotransmitter synthesis. This effect is dependent on protein synthesis and is induced after 12 hr of NMDA or KCl stimulation. Kinetics of PAG activity showed that Km values were unaffected, in contrast to Vmax values that were increased approximately 70% and 215% over control by NMDA and KCl treatment, respectively. For GLDH, we found two isoforms that were affected differentially by the experimental conditions. Western blot analysis clearly evidenced an increase of approximately 120–180% in the amount of PAG in NMDA‐ and KCl‐treated cells, whereas GLDH was not significantly modified. These results demonstrate that the NMDA‐ and KCl‐induced activation of PAG are not due to the modification of the preexisting enzyme, but to an increase in the synthesis of this enzyme. This suggests that NMDA receptor stimulation during critical periods of the cerebellar granule cell development leads to the activation of gene expression involved in the process of cell differentiation.

Leishmania mexicana lipophosphoglycan differentially regulates PKCα‐induced oxidative burst in macrophages of BALB/c and C57BL/6 mice

Leishmania are protozoan parasites that infect macrophages and their survival is partially achieved through inhibition of the cellular oxidative burst by parasite lipophosphoglycan (LPG). PKCα is the predominant PKC isoenzyme required for macrophage oxidative burst, yet it is not known if different susceptibility of BALB/c and C57BL/6 mice to Leishmania mexicana could be related to PKCα. We analysed the effect of L. mexicana promastigotes and parasite LPG on expression of PKCα and on its activity in macrophages of both mouse strains. Our data show that expression of the isoenzyme was not altered either by LPG or by L. mexicana promastigotes. Yet LPG exerted opposing effects on PKCα activity of macrophages between both strains: in susceptible BALB/c cells, it inhibited PKCα activity, whereas in the more resistant strain it augmented enzymatic activity 2·8 times. In addition, LPG inhibited oxidative burst only in susceptible BALB/c macrophages and the degree of inhibition correlated with parasite survival. Promastigotes also inhibited PKCα activity and oxidative burst in macrophages of BALB/c mice, whereas in C57BL/6, they enhanced PKCα activity and oxidative burst inhibition was less severe. Our data indicate that control of PKCα‐induced oxidative burst by L. mexicana LPG relates with its success to infect murine macrophages.

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.