Intracellular Survival of Leishmania major Depends on Uptake and Degradation of Extracellular Matrix Glycosaminoglycans by Macrophages

Leishmania parasites replicate within the phagolysosome compartment of mammalian macrophages. Although Leishmania depend on sugars as a major carbon source during infections, the nutrient composition of the phagolysosome remains poorly described. To determine the origin of the sugar carbon source in macrophage phagolysosomes, we have generated a N-acetylglucosamine acetyltransferase (GNAT) deficient Leishmania major mutant (∆gnat) that is auxotrophic for the amino sugar, N-acetylglucosamine (GlcNAc). This mutant was unable to grow or survive in ex vivo infected macrophages even when macrophages were cultivated in presence of exogenous GlcNAc. In contrast, the L. major ∆gnat mutant induced normal skin lesions in mice, suggesting that these parasites have access to GlcNAc in tissue macrophages. Intracellular growth of the mutant in ex vivo infected macrophages was restored by supplementation of the macrophage medium with hyaluronan, a GlcNAc-rich extracellular matrix glycosaminoglycan. Hyaluronan is present and constitutively turned-over in Leishmania-induced skin lesions and is efficiently internalized into Leishmania containing phagolysosomes. These findings suggest that the constitutive internalization and degradation of host glycosaminoglycans by macrophages provides Leishmania with essential carbon sources, creating a uniquely favorable niche for these parasites.     

Dolabelladienetriol, a Compound from Dictyota pfaffii Algae, Inhibits the Infection by Leishmania amazonensis

Background

Chemotherapy for leishmaniasis, a disease caused by Leishmania parasites, is expensive and causes side effects. Furthermore, parasite resistance constitutes an increasing problem, and new drugs against this disease are needed. In this study, we examine the effect of the compound 8,10,18-trihydroxy-2,6-dolabelladiene (Dolabelladienetriol), on Leishmania growth in macrophages. The ability of this compound to modulate macrophage function is also described.

Methodology/Principal Findings

Leishmania-infected macrophages were treated with Dolabelladienetriol, and parasite growth was measured using an infectivity index. Nitric oxide (NO), TNF-α and TGF-β production were assayed in macrophages using specific assays. NF-kB nuclear translocation was analyzed by western blot. Dolabelladienetriol inhibited Leishmania in a dose-dependent manner; the IC₅₀ was 44 µM. Dolabelladienetriol diminished NO, TNF-α and TGF-β production in uninfected and Leishmania-infected macrophages and reduced NF-kB nuclear translocation. Dolabelladienetriol inhibited Leishmania infection even when the parasite growth was exacerbated by either IL-10 or TGF-β. In addition, Dolabelladienetriol inhibited Leishmania growth in HIV-1-co-infected human macrophages.

Conclusion

Our results indicate that Dolabelladienetriol significantly inhibits Leishmania in macrophages even in the presence of factors that exacerbate parasite growth, such as IL-10, TGF-β and HIV-1 co-infection. Our results suggest that Dolabelladienetriol is a promising candidate for future studies regarding treatment of leishmaniasis, associated or not with HIV-1 infection.     

Leishmania infection inhibits cycloheximide-induced macrophage apoptosis in a strain-dependent manner

Activation of apoptosis is one of the most ancient mechanisms to eliminate intracellular infections; the capacity to subvert this programed cell death provides an adaptive advantage to pathogens that persist in an intracellular environment. Leishmania species are obligate intracellular parasites that primarily reside within host macrophages. We demonstrate here that Leishmania infection protects macrophages from cycloheximide-induced apoptosis in a species and strain specific manner. Our data further reveal that Leishmania phosphoglycans and direct contact between parasites and host cells are required for the inhibitory phenotype.     

Dok proteins are recruited to the phagosome and degraded in a GP63-dependent manner during Leishmania major infection

Three adaptor molecules of the Dok family, Dok-1, Dok-2 and Dok-3 are expressed in macrophages and are involved in the negative regulation of signaling in response to lipopolysaccharide and various cytokines and growth factors. We investigated the role and the fate of these proteins following infection with Leishmania major promastigotes in macrophages. The protozoan parasite L. major causes cutaneous leishmaniasis and is known for its capacity to alter host-cell signaling and function. Dok-1/Dok-2^−/− bone marrow-derived macrophages displayed normal uptake of L. major promastigotes. Following Leishmania infection, Dok-1 was barely detectable by confocal microscopy. By contrast, phagocytosis of latex beads or zymosan led to the recruitment of Dok-1 to phagosomes. In the absence of the Leishmania pathogenesis-associated metalloprotease GP63, Dok-1 was also, partially, recruited to phagosomes containing L. major promastigotes. Further biochemical analyses revealed that similar to Dok-1, Dok-2 and Dok-3 were targets of GP63. Moreover, we showed that upon infection with wild-type or Δgp63 L. major promastigotes, production of nitric oxide and tumor necrosis factor by interferon-γ-primed Dok-1/Dok-2^−/− macrophages was reduced compared to WT macrophages. These results suggest that Dok proteins may be important regulators of macrophage responses to Leishmania infection.     

Essential role of leukotriene B4 on Leishmania (Viannia) braziliensis killing by human macrophages

Although Leishmania (Viannia) braziliensis is the most prevalent species that cause American tegumentary leishmaniasis (ATL), the immune response against this parasite has been poorly investigated. Upon activation, macrophages produce a series of pro-inflammatory molecules, including the lipid mediator leukotriene B₄ (LTB₄). LTB₄ has been shown to enhance several macrophage functions, but its role in human macrophages is less known. Here, we investigated the role of LTB₄ on human monocyte-derived macrophages infected with human isolate of L. (V.) braziliensis (IMG3). It was found that human macrophages produce LTB₄ upon infection with Leishmania, which by autocrine or paracrine activation of its high affinity receptor BLT1, potentiates macrophage leishmanicidal activity. This LTB₄ effect is mediated by increased secretion of reactive oxygen species (ROS). Moreover, Leishmania infection decreased the expression of BLT1, leading to the speculation that this could represent a parasite escape mechanism to establish a chronic inflammatory infection. Therefore, our data suggest that LTB₄ could be used in therapeutic strategies to control Leishmania infection.     

Integrity of the Actin Cytoskeleton of Host Macrophages is Essential for Leishmania donovani Infection

Visceral leishmaniasis is a vector-borne disease caused by an obligate intracellular protozoan parasite Leishmania donovani. The molecular mechanism involved in internalization of Leishmania is poorly understood. The entry of Leishmania involves interaction with the plasma membrane of host cells. We have previously demonstrated the requirement of host membrane cholesterol in the binding and internalization of L. donovani into macrophages. In the present work, we explored the role of the host actin cytoskeleton in leishmanial infection. We observed a dose-dependent reduction in the attachment of Leishmania promastigotes to host macrophages upon destabilization of the actin cytoskeleton by cytochalasin D. This is accompanied by a concomitant reduction in the intracellular amastigote load. We utilized a recently developed high resolution microscopy-based method to quantitate cellular F-actin content upon treatment with cytochalasin D. A striking feature of our results is that binding of Leishmania promastigotes and intracellular amastigote load show close correlation with cellular F-actin level. Importantly, the binding of Escherichia coli remained invariant upon actin destabilization of host cells, thereby implying specific involvement of the actin cytoskeleton in Leishmania infection. To the best of our knowledge, these novel results constitute the first comprehensive demonstration on the specific role of the host actin cytoskeleton in Leishmania infection. Our results could be significant in developing future therapeutic strategies to tackle leishmaniasis.     

Amphotericin B inhibits entry of Leishmania donovani into primary macrophages

Visceral leishmaniasis is a vector-borne disease caused by an obligate intra-macrophage protozoan parasite Leishmania donovani. The molecular mechanisms involved in internalization of Leishmania are still poorly understood. Amphotericin B and its formulations are considered as the best existing drugs against visceral leishmaniasis and are being increasingly used. The reason for its antileishmanial activity is believed to be its ability to bind ergosterol found in parasite membranes. In case of in vivo amphotericin B treatment, both host macrophages and parasites are exposed to amphotericin B. The effect of amphotericin B treatment could therefore be due to a combination of its interaction with both sterols i.e., ergosterol of Leishmania and cholesterol of host macrophages. We report here that cholesterol complexation by amphotericin B markedly inhibits binding of L. donovani promastigotes to macrophages. These results represent one of the first reports on the effect of amphotericin B on the binding of Leishmania parasites to host macrophages. Importantly, these results offer the possibility of reevaluating the mechanism behind the effectiveness of current therapeutic strategies that employ sterol-complexing agents such as amphotericin B to treat leishmaniasis.     

Intracellular iron availability modulates the requirement for Leishmania Iron Regulator 1 (LIR1) during macrophage infections

The Leishmania plasma membrane transporter Leishmania Iron Regulator 1 (LIR1) facilitates iron export and is required for parasite virulence. By modulating macrophage iron content, we investigated the host site where LIR1 regulates Leishmania amazonensis infectivity. In bone marrow-derived macrophages, LIR1 null mutants demonstrated a paradoxical increase in virulence during infections in heme-depleted media, while wild-type growth was inhibited under the same conditions. Loading the endocytic pathway of macrophages with cationized ferritin prior to infection reversed the effect of heme depletion on both strains. Thus, LIR1 contributes to Leishmania virulence by protecting the parasites from toxicity resulting from iron accumulation inside parasitophorous vacuoles.     

Trypanosome Lytic Factor,an Antimicrobial High-Density Lipoprotein,Ameliorates Leishmania Infection

Innate immunity is the first line of defense against invading microorganisms. Trypanosome Lytic Factor (TLF) is a minor sub-fraction of human high-density lipoprotein that provides innate immunity by completely protecting humans from infection by most species of African trypanosomes, which belong to the Kinetoplastida order. Herein, we demonstrate the broader protective effects of human TLF, which inhibits intracellular infection by Leishmania, a kinetoplastid that replicates in phagolysosomes of macrophages. We show that TLF accumulates within the parasitophorous vacuole of macrophages in vitro and reduces the number of Leishmania metacyclic promastigotes, but not amastigotes. We do not detect any activation of the macrophages by TLF in the presence or absence of Leishmania, and therefore propose that TLF directly damages the parasite in the acidic parasitophorous vacuole. To investigate the physiological relevance of this observation, we have reconstituted lytic activity in vivo by generating mice that express the two main protein components of TLFs: human apolipoprotein L-I and haptoglobin-related protein. Both proteins are expressed in mice at levels equivalent to those found in humans and circulate within high-density lipoproteins. We find that TLF mice can ameliorate an infection with Leishmania by significantly reducing the pathogen burden. In contrast, TLF mice were not protected against infection by the kinetoplastid Trypanosoma cruzi, which infects many cell types and transiently passes through a phagolysosome. We conclude that TLF not only determines species specificity for African trypanosomes, but can also ameliorate an infection with Leishmania, while having no effect on T. cruzi. We propose that TLFs are a component of the innate immune system that can limit infections by their ability to selectively damage pathogens in phagolysosomes within the reticuloendothelial system.     

Impact of Neutrophil-Secreted Myeloid Related Proteins 8 and 14 (MRP 8/14) on Leishmaniasis Progression

The myeloid-related proteins (MRPs) 8/14 are small proteins mainly produced by neutrophils, which have been reported to induce NO production in macrophages. On the other hand, Leishmania survives and multiplies within phagocytes by inactivating several of their microbicidal functions. Whereas MRPs are rapidly released during the innate immune response, their role in the regulation of Leishmaniasis is still unknown. In vitro experiments revealed that Leishmania infection alters MRP-induced signaling, leading to inhibition of macrophage functions (NO, TNF-α). In contrast, MRP-primed cells showed normal signaling activation and NO production in response to Leishmania infection. Using a murine air-pouch model, we observed that infection with L. major induced leukocyte recruitment and MRP secretion comparable to LPS-treated mice. Depletion of MRPs significantly reduced these inflammatory events and augmented both parasite load and footpad swelling during the first 8 weeks post-infection, as also observed in MRP KO mice. On the contrary, mouse treatment with recombinant MRPs (rMRPs) had the opposite effect. Collectively, our results suggest that rapid secretion of MRPs by neutrophils at the site of infection may protect uninfected macrophages and favor a more efficient innate inflammatory response against Leishmania infection. In summary, our study reveals the critical role played by MRPs in the regulation of Leishmania infection and how this pathogen can subvert its action.     

Leishmania (Viannia) panamensis: An in vitro assay using the expression of GFP for screening of antileishmanial drug

Promastigotes of Leishmania (Viannia) panamensis were successfully transfected with p6.5-egfp to express green fluorescent protein. The transfectants remained infective to macrophages, providing an in vitro model for screening antileishmanial drugs. This was demonstrated by flow cytometry of macrophage-associated GFP after exposure of infected cultures to known antileishmanial drugs, i.e. amphotericin B and glucantime^®. Fluorescence of GFP diminished progressively from infected cells with increasing drug concentrations used in both cases. The availability of this fluorescent assay for infection of macrophages by L. (V.) panamensis facilitates drug discovery program for the Viannia species, which differ significantly from those of the Leishmania subgenus.     

PKC/ROS-Mediated NLRP3 Inflammasome Activation Is Attenuated by Leishmania Zinc-Metalloprotease during Infection

Parasites of the Leishmania genus infect and survive within macrophages by inhibiting several microbicidal molecules, such as nitric oxide and pro-inflammatory cytokines. In this context, various species of Leishmania have been reported to inhibit or reduce the production of IL-1β both in vitro and in vivo. However, the mechanism whereby Leishmania parasites are able to affect IL-1β production and secretion by macrophages is still not fully understood. Dependent on the stimulus at hand, the maturation of IL-1β is facilitated by different inflammasome complexes. The NLRP3 inflammasome has been shown to be of pivotal importance in the detection of danger molecules such as inorganic crystals like asbestos, silica and malarial hemozoin, (HZ) as well as infectious agents. In the present work, we investigated whether Leishmania parasites modulate NLRP3 inflammasome activation. Using PMA-differentiated THP-1 cells, we demonstrate that Leishmania infection effectively inhibits macrophage IL-1β production upon stimulation. In this context, the expression and activity of the metalloprotease GP63 - a critical virulence factor expressed by all infectious Leishmania species - is a prerequisite for a Leishmania-mediated reduction of IL-1β secretion. Accordingly, L. mexicana, purified GP63 and GP63-containing exosomes, caused the inhibition of macrophage IL-1β production. Leishmania-dependent suppression of IL-1β secretion is accompanied by an inhibition of reactive oxygen species (ROS) production that has previously been shown to be associated with NLRP3 inflammasome activation. The observed loss of ROS production was due to an impaired PKC-mediated protein phosphorylation. Furthermore, ROS-independent inflammasome activation was inhibited, possibly due to an observed GP63-dependent cleavage of inflammasome and inflammasome-related proteins. Collectively for the first time, we herein provide evidence that the protozoan parasite Leishmania, through its surface metalloprotease GP63, can significantly inhibit NLRP3 inflammasome function and IL-1β production.     

Selective Fusion of Azurophilic Granules with Leishmania-containing Phagosomes in Human Neutrophils

Leishmania parasites use polymorphonuclear neutrophils as intermediate hosts before their ultimate delivery to macrophages following engulfment of parasite-infected neutrophils. This leads to a silent and unrecognized entry of Leishmania into the macrophage host cell. Neutrophil function depends on its cytoplasmic granules, but their mobilization and role in how Leishmania parasites evade intracellular killing in neutrophils remain undetermined. Here, we have found by ultrastructural approaches that neutrophils ingested Leishmania major promastigotes, and azurophilic granules fused in a preferential way with parasite-containing phagosomes, without promoting parasite killing. Azurophilic granules, identified by the granule marker myeloperoxidase, also fused with Leishmania donovani-engulfed vacuoles in human neutrophils. In addition, the azurophilic membrane marker CD63 was also detected in the vacuole surrounding the parasite, and in the fusion of azurophilic granules with the parasite-engulfed phagosome. Tertiary and specific granules, involved in vacuole acidification and superoxide anion generation, hardly fused with Leishmania-containing phagosomes. L. major interaction with neutrophils did not elicit production of reactive oxygen species or mobilization of tertiary and specific granules. By using immunogold electron microscopy approaches in the engulfment of L. major and L. donovani by human neutrophils, we did not find a significant contribution of endoplasmic reticulum to the formation of Leishmania-containing vacuoles. Live Leishmania parasites were required to be optimally internalized by neutrophils. Our data suggest that Leishmania promastigotes modulate their uptake by neutrophils, and regulate granule fusion processes in a rather selective way to favor parasite survival in human neutrophils.     

Bidirectional cytokine-microRNA control: A novel immunoregulatory framework in leishmaniasis

As effector innate immune cells and as a host to the protozoan parasite Leishmania, macrophages play a dual role in antileishmanial immunoregulation. The 2 key players in this immunoregulation are the macrophage-expressed microRNAs (miRNAs) and the macrophage-secreted cytokines. miRNAs, as small noncoding RNAs, play vital roles in macrophage functions including cytokines and chemokines production. In the reverse direction, Leishmania-regulated cytokines alter miRNAs expression to regulate the antileishmanial functions of macrophages. The miRNA patterns vary with the time and stage of infection. The cytokine-regulated macrophage miRNAs not only help parasite elimination or persistence but also regulate cytokine production from macrophages. Based on these observations, we propose a novel immunoregulatory framework as a scientific rationale for antileishmanial therapy.