Characterization of Argonaute-containing protein complexes in Leishmania-infected human macrophages.

The intracellular protozoan parasite Leishmania causes leishmaniasis in humans, leading to serious illness and death in tropical and subtropical areas worldwide. Unfortunately, due to the unavailability of approved vaccines for humans and the limited efficacy of available drugs, leishmaniasis is on the rise. A comprehensive understanding of host-pathogen interactions at the molecular level could pave the way to counter leishmaniasis. There is growing evidence that several intracellular pathogens target RNA interference (RNAi) pathways in host cells to facilitate their persistence. The core elements of the RNAi system are complexes of Argonaute (Ago) proteins with small non-coding RNAs, also known as RNA-induced silencing complexes (RISCs). Recently, we have shown that Leishmania modulates Ago1 protein of host macrophages for its survival. In this study, we biochemically characterize the Ago proteins' interactome in Leishmania-infected macrophages compared to non-infected cells. For this, a quantitative proteomic approach using stable isotope labelling by amino acids in cell culture (SILAC) was employed, followed by purification of host Ago-complexes using a short TNRC6 protein-derived peptide fused to glutathione S-transferase beads as an affinity matrix. Proteomic-based detailed biochemical analysis revealed Leishmania modulated host macrophage RISC composition during infection. This analysis identified 51 Ago-interacting proteins with a broad range of biological activities. Strikingly, Leishmania proteins were detected as part of host Ago-containing complexes in infected cells. Our results present the first report of comprehensive quantitative proteomics of Ago-containing complexes isolated from Leishmania-infected macrophages and suggest targeting the effector complex of host RNAi machinery. Additionally, these results expand knowledge of RISC in the context of host-pathogen interactions in parasitology in general.

Targeting Trypanothione Metabolism in Trypanosomatids.

Infectious diseases caused by trypanosomatids, including African trypanosomiasis (sleeping sickness), Chagas disease, and different forms of leishmaniasis, are Neglected Tropical Diseases affecting millions of people worldwide, mainly in vulnerable territories of tropical and subtropical areas. In general, current treatments against these diseases are old-fashioned, showing adverse effects and loss of efficacy due to misuse or overuse, thus leading to the emergence of resistance. For these reasons, searching for new antitrypanosomatid drugs has become an urgent necessity, and different metabolic pathways have been studied as potential drug targets against these parasites. Considering that trypanosomatids possess a unique redox pathway based on the trypanothione molecule absent in the mammalian host, the key enzymes involved in trypanothione metabolism, trypanothione reductase and trypanothione synthetase, have been studied in detail as druggable targets. In this review, we summarize some of the recent findings on the molecules inhibiting these two essential enzymes for Trypanosoma and Leishmania viability.

Leishmania infection upregulates and engages host macrophage Argonaute 1, and system-wide proteomics reveals Argonaute 1-dependent host response.

Leishmania donovani, an intracellular protozoan parasite, is the causative agent of visceral leishmaniasis, the most severe form of leishmaniasis in humans. It is becoming increasingly clear that several intracellular pathogens target host cell RNA interference (RNAi) pathways to promote their survival. Complexes of Argonaute proteins with small RNAs are core components of the RNAi. In this study, we investigated the potential role of host macrophage Argonautes in Leishmania pathogenesis. Using Western blot analysis of Leishmania donovani-infected macrophages, we show here that Leishmania infection selectively increased the abundance of host Argonaute 1 (Ago1). This increased abundance of Ago1 in infected cells also resulted in higher levels of Ago1 in active Ago-complexes, suggesting the preferred use of Ago1 in RNAi in Leishmania-infected cells. This analysis used a short trinucleotide repeat containing 6 (TNRC6)/glycine-tryptophan repeat protein (GW182) protein-derived peptide fused to Glutathione S-transferase as an affinity matrix to capture mature Ago-small RNAs complexes from the cytosol of non-infected and Leishmania-infected cells. Furthermore, Ago1 silencing significantly reduced intracellular survival of Leishmania, demonstrating that Ago1 is essential for Leishmania pathogenesis. To investigate the role of host Ago1 in Leishmania pathogenesis, a quantitative whole proteome approach was employed, which showed that expression of several previously reported Leishmania pathogenesis-related proteins was dependent on the level of macrophage Ago1. Together, these findings identify Ago1 as the preferred Argonaute of RNAi machinery in infected cells and a novel and essential virulence factor by proxy that promotes Leishmania survival.

Proteomic research on new urinary biomarkers of renal disease in canine leishmaniosis: Survival and monitoring response to treatment.

The objective of our study was to search for survival biomarkers (SB) and treatment response monitoring biomarkers (TRMB) in the urinary proteome of dogs with renal disease secondary to canine leishmaniosis (CanL), using UHPLC-MS/MS. The proteomic data are available via ProteomeXchange with identifier PXD042578. Initially, a group of 12 dogs was evaluated and divided into survivors (SG; n = 6) and nonsurvivors (NSG; n = 6). A total of 972 proteins were obtained from the evaluated samples. Then, bioinformatic analysis reduced them to 6 proteins like potential SB increased in the NSG, specifically, Haemoglobin subunit Alpha 1, Complement Factor I, Complement C5, Fibrinogen beta chain (fragment), Peptidase S1 domain-containing protein, and Fibrinogen gamma chain. Afterwards, SG was used to search for TRMB, studying their urine at 0, 30, and 90 days, and 9 proteins that decreased after treatment were obtained: Apolipoprotein E, Cathepsin B, Cystatin B, Cystatin-C-like, Lysozyme, Monocyte differentiation CD14, Pancreatitis-associated precursor protein, Profilin, and Protein FAM3C. Finally, enrichment analysis provided information about the biological mechanisms in which these proteins are involved. In conclusion, this study provides 15 new candidate urinary biomarkers and an improved understanding of the pathogenesis of kidney disease in CanL.

Unwinding the mechanism of macrophage repolarization potential of Oceanimonas sp. BPMS22-derived protein protease inhibitor through Toll-like receptor 4 against experimental visceral leishmaniasis.

The Oceanimonas sp. BPMS22-derived protein protease inhibitor (PPI) has been proven to shift macrophages towards an inflammatory state and reduce Leishmania donovani infection in vitro and in vivo. The current study explored and validated the mechanistic aspects of the PPI and Toll-like receptor (TLR) interaction. The PPI exhibited the upregulation of TLR2, TLR4, and TLR6 during treatment which was proven to orchestrate parasite clearance effectively. An in silico study confirmed the high interaction with TLR4 and PPI. Immune blotting confirmed the significant upregulation of TLR4 in macrophages irrespective of L. donovani infection. Pharmacological inhibition and immune blot study confirmed the involvement of the PPI in TLR4-mediated phosphorylation of p38 MAPK and dephosphorylation of ERK1/2, repolarizing to pro-inflammatory macrophage state against experimental visceral leishmaniasis. In addition, in TLR4 knockdown condition, PPI treatment failed to diminish M2 phenotypical markers (CD68, Fizz1, Ym1, CD206, and MSR-2) and anti-inflammatory cytokines (IL-4, IL-10, and TGF-ß). Simultaneously, the PPI failed to upregulate the M1 phenotypical markers and pro-inflammatory cytokines (IL-1ß, IL-6, IL-12, and IFN-γ) (p < 0.001) during the TLR4 knockdown condition. In the absence of TLR4, the PPI also failed to reduce the parasite load and T-cell proliferation and impaired the delayed-type hypersensitivity response. The absence of pro-inflammatory cytokines was observed during a co-culture study with PPI-treated macrophages (in the TLR4 knockdown condition) with day 10 T-cell obtained from L. donovani-infected mice. This study supports the immunotherapeutic potential of the PPI as it interacted with TLR4 and promoted macrophage repolarization (M2-M1) to restrict the L. donovani parasite burden and helps in the mounting immune response against experimental visceral leishmaniasis.

Leishmania infection-derived extracellular vesicles drive transcription of genes involved in M2 polarization.

Although it is known that the composition of extracellular vesicles (EVs) is determined by the characteristics of the cell and its environment, the effects of intracellular infection on EV composition and functions are not well understood. We had previously shown that cultured macrophages infected with Leishmania parasites release EVs (LiEVs) containing parasite-derived molecules. In this study we show that LdVash, a molecule previously identified in LiEVs from L. donovani infected RAW264.7 macrophages, is widely distributed in the liver of L. donovani infected mice. This result shows for the first time that parasite molecules are released in EVs and distributed in infected tissues where they can be endocytosed by cells in the liver, including macrophages that significantly increase numbers as the infection progresses. To evaluate the potential impact of LiEVs on macrophage functions, we show that primary peritoneal exudate macrophages (PECs) express transcripts of signature molecules of M2 macrophages such as arginase 1, IL-10, and IL-4R when incubated with LiEVs. In comparative studies that illustrate how intracellular pathogens control the composition and functions of EVs released from macrophages, we show that EVs from RAW264.7 macrophages infected with Salmonella Typhimurium activate PECs to express transcripts of signature molecules of M1 macrophages such as iNOS, TNF alpha, and IFN-gamma and not M2 signature molecules. Finally, in contrast to the polarized responses observed in in vitro studies of macrophages, both M1 and M2 signature molecules are detected in L. donovani infected livers, although they exhibit differences in their spatial distribution in infected tissues. In conclusion, EVs produced by macrophages during Leishmania infection lead to the gene expression consistent with M2 polarization. In contrast, the EVs produced during S. Typhimurium infection stimulated the transcription of genes associated with M1 polarization.

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.

A novel leishmanial copper P-type ATPase plays a vital role in parasite infection and intracellular survival.

Copper (Cu) is essential for all life forms; however, in excess, it becomes toxic. Toxic properties of Cu are known to be utilized by host species against various pathogenic invasions. Leishmania, in both free-living and intracellular forms, exhibits appreciable tolerance toward Cu stress. While determining the mechanism of Cu-stress evasion employed by Leishmania, we identified and characterized a hitherto unknown Cu-ATPase in Leishmania major and established its role in parasite survival in host macrophages. This novel L. major Cu-ATPase, LmATP7, exhibits homology with its orthologs at multiple motifs. In promastigotes, LmATP7 primarily localized at the plasma membrane. We also show that LmATP7 exhibits Cu-dependent expression patterns and complements Cu transport in a Cu-ATPase-deficient yeast strain. Promastigotes overexpressing LmATP7 exhibited higher survival upon Cu stress, indicating efficacious Cu export compared with Wt and heterozygous LmATP7 knockout parasites. We further explored macrophage-Leishmania interactions with respect to Cu stress. We found that Leishmania infection triggers upregulation of major mammalian Cu exporter, ATP7A, in macrophages, and trafficking of ATP7A from the trans-Golgi network to endolysosomes in macrophages harboring amastigotes. Simultaneously, in Leishmania, we observed a multifold increase in LmATP7 transcripts as the promastigote becomes established in macrophages and morphs to the amastigote form. Finally, overexpressing LmATP7 in parasites increases amastigote survivability within macrophages, whereas knocking it down reduces survivability drastically. Mice injected in their footpads with an LmATP7-overexpressing strain showed significantly larger lesions and higher amastigote loads as compared with controls and knockouts. These data establish the role of LmATP7 in parasite infectivity and intramacrophagic survivability.

Variation in Leishmania chemokine suppression driven by diversification of the GP63 virulence factor.

Leishmaniasis is a neglected tropical disease with diverse outcomes ranging from self-healing lesions, to progressive non-healing lesions, to metastatic spread and destruction of mucous membranes. Although resolution of cutaneous leishmaniasis is a classic example of type-1 immunity leading to self-healing lesions, an excess of type-1 related inflammation can contribute to immunopathology and metastatic spread. Leishmania genetic diversity can contribute to variation in polarization and robustness of the immune response through differences in both pathogen sensing by the host and immune evasion by the parasite. In this study, we observed a difference in parasite chemokine suppression between the Leishmania (L.) subgenus and the Viannia (V.) subgenus, which is associated with severe immune-mediated pathology such as mucocutaneous leishmaniasis. While Leishmania (L.) subgenus parasites utilize the virulence factor and metalloprotease glycoprotein-63 (gp63) to suppress the type-1 associated host chemokine CXCL10, L. (V.) panamensis did not suppress CXCL10. To understand the molecular basis for the inter-species variation in chemokine suppression, we used in silico modeling to identify a putative CXCL10-binding site on GP63. The putative CXCL10 binding site is in a region of gp63 under significant positive selection, and it varies from the L. major wild-type sequence in all gp63 alleles identified in the L. (V.) panamensis reference genome. Mutating wild-type L. (L.) major gp63 to the L. (V.) panamensis sequence at the putative binding site impaired cleavage of CXCL10 but not a non-specific protease substrate. Notably, Viannia clinical isolates confirmed that L. (V.) panamensis primarily encodes non-CXCL10-cleaving gp63 alleles. In contrast, L. (V.) braziliensis has an intermediate level of activity, consistent with this species having more equal proportions of both alleles. Our results demonstrate how parasite genetic diversity can contribute to variation in immune responses to Leishmania spp. infection that may play critical roles in the outcome of infection.

Activity of Anti-Microbial Peptides (AMPs) against Leishmania and Other Parasites: An Overview.

Anti-microbial peptides (AMPs), small biologically active molecules, produced by different organisms through their innate immune system, have become a considerable subject of interest in the request of novel therapeutics. Most of these peptides are cationic-amphipathic, exhibiting two main mechanisms of action, direct lysis and by modulating the immunity. The most commonly reported activity of AMPs is their anti-bacterial effects, although other effects, such as anti-fungal, anti-viral, and anti-parasitic, as well as anti-tumor mechanisms of action have also been described. Their anti-parasitic effect against leishmaniasis has been studied. Leishmaniasis is a neglected tropical disease. Currently among parasitic diseases, it is the second most threating illness after malaria. Clinical treatments, mainly antimonial derivatives, are related to drug resistance and some undesirable effects. Therefore, the development of new therapeutic agents has become a priority, and AMPs constitute a promising alternative. In this work, we describe the principal families of AMPs (melittin, cecropin, cathelicidin, defensin, magainin, temporin, dermaseptin, eumenitin, and histatin) exhibiting a potential anti-leishmanial activity, as well as their effectiveness against other microorganisms.

Metabolomic Reprogramming of C57BL/6-Macrophages during Early Infection with L. amazonensis.

Leishmania survival inside macrophages depends on factors that lead to the immune response evasion during the infection. In this context, the metabolic scenario of the host cell-parasite relationship can be crucial to understanding how this parasite can survive inside host cells due to the host's metabolic pathways reprogramming. In this work, we aimed to analyze metabolic networks of bone marrow-derived macrophages from C57BL/6 mice infected with Leishmania amazonensis wild type (La-WT) or arginase knocked out (La-arg-), using the untargeted Capillary Electrophoresis-Mass Spectrometry (CE-MS) approach to assess metabolomic profile. Macrophages showed specific changes in metabolite abundance upon Leishmania infection, as well as in the absence of parasite-arginase. The absence of L. amazonensis-arginase promoted the regulation of both host and parasite urea cycle, glycine and serine metabolism, ammonia recycling, metabolism of arginine, proline, aspartate, glutamate, spermidine, spermine, methylhistidine, and glutathione metabolism. The increased L-arginine, L-citrulline, L-glutamine, oxidized glutathione, S-adenosylmethionine, N-acetylspermidine, trypanothione disulfide, and trypanothione levels were observed in La-WT-infected C57BL/6-macrophage compared to uninfected. The absence of parasite arginase increased L-arginine, argininic acid, and citrulline levels and reduced ornithine, putrescine, S-adenosylmethionine, glutamic acid, proline, N-glutamyl-alanine, glutamyl-arginine, trypanothione disulfide, and trypanothione when compared to La-WT infected macrophage. Moreover, the absence of parasite arginase leads to an increase in NO production levels and a higher infectivity rate at 4 h of infection. The data presented here show a host-dependent regulation of metabolomic profiles of C57BL/6 macrophages compared to the previously observed BALB/c macrophages infected with L. amazonensis, an important fact due to the dual and contrasting macrophage phenotypes of those mice. In addition, the Leishmania-arginase showed interference with the urea cycle, glycine, and glutathione metabolism during host-pathogen interactions.

Oxidant activated soluble adenylate cyclase of Leishmania donovani regulates the cAMP-PKA signaling axis for its intra-macrophage survival during infection.

Adenosine 3',5'-cyclic monophosphate (cAMP) is a stress sensor molecule that transduces the cellular signal when Leishmania donovani moves from insect vector to mammalian host. At this stage, the parasite membrane-bound receptor adenylate cyclase predominantly produces cAMP to cope with the oxidative assault imposed by host macrophages. However, the role of soluble adenylate cyclase of L. donovani (LdHemAC) has not been investigated fully. In the present investigation, we monitored an alternative pool of cAMP, maintained by LdHemAC. The elevated cAMP effectively transmits signals by binding to Protein Kinase A (PKA) present in the cytosol and regulates antioxidant gene expression and phosphorylates several unknown PKA substrate proteins. Menadione-catalyzed production of reactive oxygen species (ROS) mimics host oxidative condition in vitro in parasites where cAMP production and PKA activity were found increased by ~1.54 ± 0.35, and ~1.78 ± 0.47-fold, respectively while expression of LdHemAC gene elevated by ~2.18 ± 0.17-fold. The LdHemAC sense these oxidants and became activated to cyclize ATP to enhance the cAMP basal level that regulates antioxidant gene expression to rescue parasites from oxidative stress. In knockdown parasites (LdHemAC-KD), the downregulated antioxidant genes expression, namely, Sod (2.30 ± 0.46), Pxn (2.73 ± 0.15), Tdr (2.7 ± 0.12), and Gss (1.57 ± 0.15) results in decreased parasite viability while in overexpressed parasites (LdHemAC-OE), the expression was upregulated by ~5.7 ± 0.35, ~2.57 ± 0.56, ~4.7 ± 0.36, and ~2.4 ± 0.83, respectively, which possibly overcomes ROS accumulation and enhances viability. Furthermore, LdHemAC-OE higher PKA activity regulates phosphorylation of substrate proteins (~56 kDs in membrane fraction and ~25 kDs in the soluble fraction). It reduced significantly when treated with inhibitors like DDA, Rp-cAMP, and H-89 and increased by ~2.1 ± 0.28-fold, respectively under oxidative conditions. The LdHemAC-KD was found less infective to RAW 264.7 macrophages and more prone to oxidative damage as compared to LdHemAC-OE and control parasites. Together, this study demonstrates mechanistic links among LdHemAC, cAMP, and PKA in parasite survival and invasion under host oxidative condition.

Clustering of adenosine A2B receptors with ectonucleotidases in caveolin-rich lipid rafts underlies immunomodulation by Leishmania amazonensis.

Extracellular adenosine plays important roles in modulating the immune responses. We have previously demonstrated that infection of dendritic cells (DC) by Leishmania amazonensis leads to increased expression of CD39 and CD73 and to the selective activation of the low affinity A2B receptors (A2B R), which contributes to DC inhibition, without involvement of the high affinity A2A R. To understand this apparent paradox, we now characterized the alterations of both adenosine receptors in infected cells. With this aim, bone marrow-derived DC from C57BL/6J mice were infected with metacyclic promastigotes of L. amazonensis. Fluorescence microscopy revealed that L. amazonensis infection stimulates the recruitment of A2B R, but not of A2A R, to the surface of infected DC, without altering the amount of mRNA or the total A2B R density, an effect dependent on lipophosphoglycan (LPG). Log-phase promastigotes or axenic amastigotes of L. amazonensis do not stimulate A2B R recruitment. A2B R clusters are localized in caveolin-rich lipid rafts and the disruption of these membrane domains impairs A2B R recruitment and activation. More importantly, our results show that A2B R co-localize with CD39 and CD73 forming a "purinergic cluster" that allows for the production of extracellular adenosine in close proximity with these receptors. We conclude that A2B R activation by locally produced adenosine constitutes an elegant and powerful evasion mechanism used by L. amazonensis to down-modulate the DC activation.

Eosinophils increase macrophage ability to control intracellular Leishmania amazonensis infection via PGD2 paracrine activity in vitro.

Clinical and experimental studies have described eosinophil infiltration in Leishmania amazonensis infection sites, positioning eosinophils strategically adjacent to the protozoan-infected macrophages in cutaneous leishmaniasis. Here, by co-culturing mouse eosinophils with L. amazonensis-infected macrophages, we studied the impact of eosinophils on macrophage ability to regulate intracellular L. amazonensis infection. Eosinophils prevented the increase in amastigote numbers within macrophages by a mechanism dependent on a paracrine activity mediated by eosinophil-derived prostaglandin (PG) D2 acting on DP2 receptors. Exogenous PGD2 mimicked eosinophil-mediated effect on managing L. amazonensis intracellular infection by macrophages and therefore may function as a complementary tool for therapeutic intervention in L. amazonensis-driven cutaneous leishmaniasis.

A "Golden Age" for the discovery of new antileishmanial agents: Current status of leishmanicidal gold complexes and prospective targets beyond the trypanothione system.

Leishmaniasis is one of the most neglected diseases worldwide and is considered a serious public health issue. The current therapeutic options have several disadvantages that make the search for new therapeutics urgent. Gold compounds are emerging as promising candidates based on encouraging in vitro and limited in vivo results for several AuI and AuIII complexes. The antiparasitic mechanisms of these molecules remain only partially understood. However, a few studies have proposed the trypanothione redox system as a target, similar to the mammalian thioredoxin system, pointed out as the main target for several gold compounds with significant antitumor activity. In this review, we present the current status of the investigation and design of gold compounds directed at treating leishmaniasis. In addition, we explore potential targets in Leishmania parasites beyond the trypanothione system, taking into account previous studies and structure modulation performed for gold-based compounds.

Super enhancer-mediated transcription of miR146a-5p drives M2 polarization during Leishmania donovani infection.

The outcome of Leishmania donovani infection depends upon the dynamic interchanges between M1 and M2 macrophages. Information of the involvement of microRNAs (miRNAs) and epigenetic modifiers in regulating macrophage plasticity during L. donovani infection is still elusive. Differential expression analysis of polarization-regulating miRNAs, revealed significant enrichment of miR146a-5p during Leishmania donovani infection. A sustained enrichment of miR146a-5p was observed in both infected bone marrow derived macrophages (BMDMs) and BALB/c mice organs. We found involvement of miR146a-5p in phagocytosis and survivability of parasites. Moreover, miR146a-5pgot enriched in interleukin 4- stimulated BMDMs, indicating its possible involvement in M2 polarization. Upon transfecting BMDMs with miRVANA anti-146a oligos, M2 markers (CCR7, YM-1, FIZZ-1, arginase-1, IL10 and IL4) and transcription factors (p-STAT6 and c/EBPß) got depleted with concomitant augmentation of M1-polarizing transcription factors (p-STAT1, AP1 and IRF-1), miR146a target genes (TRAF6 and IRAK1), M1 cytokines (IL12 and TNFα), iNOS, nitric oxide, and nuclear translocation of phospho p-65 subunit. Neutralization of intracellular mature miR146a-5p pool in infected BALB/c mice lower organ parasite burden and expressions of M2 markers and IL10 with enrichment of M1 markers like iNOS and IL12. Additionally, we explored the novel role of super enhancer (SE), a cis-acting regulatory component, to enrich miR146a-5p expression during infection. Enhanced expression and nuclear retention of SE components like BET bromodomain 4 (BRD4) and p300 were found in infected BMDMs. Upon silencing BRD4, expressions of miR146a-5p and M2 markers were down regulated and TRAF6, IRAK1 and iNOS levels increased. STRING V.11 based predication and immune precipitation confirmed the strong interaction amongst BRD4, p300 and RNA pol II (RpbI). Chromatin immune precipitation studies suggested the recruitment of BRD4 at the enhancer loci of miR146a-5p gene during infection. Altogether, our findings revealed a novel role of BRD4/p300-depdendent super-enhancer in regulating miR146a expression during L. donovani infection which in turn mediates M2 polarization and immune-suppression.

Regulation of macrophage subsets and cytokine production in leishmaniasis.

Macrophages are host cells for parasites of the genus Leishmania where they multiply inside parasitophorous vacuoles. Paradoxically, macrophages are also the cells responsible for killing or controlling parasite growth, if appropriately activated. In this review, we will cover the patterns of macrophage activation and the mechanisms used by the parasite to circumvent being killed. We will highlight the impacts of the vector bite on macrophage activation. Finally, we will discuss the ontogeny of macrophages that are infected by Leishmania spp.

Role of thrombopoiesis in leishmaniasis.

The blood vascular system of mammals is unique in nature; inhabited with a pool of tiny small cell fragments called platelets; attributed with the most important patrolling tasks to check integrity of the entire endothelial landscape. Their production is tightly coupled with hematopoietic system where everything starts from self renewable multipotent hematopoietic stem cells (HSCs) which eventually undergo dual step (megakaryopoiesis-thrombopoiesis) thrombocytes production. Several cytokines tune the fate of every progenitor cells during hematopoiesis through temporal activation of specific transcription factors. Though platelets generated through steady state hematopoiesis are involved in the regulation of vascular homeostasis, these cells can sense pathogens through its innate immune sensors and can mount crucial responses against the invading pathogen. For this, the primary aim of many infections including Leishmania is to induce thrombocytopenia within infected host. But the underlying mechanism of this induced thrombocytopenia in Leishmania infection has not been evaluated. Elucidation of these mechanisms will be fruitful to design new chemotherapeutic strategies.

The Leishmania donovani SENP Protease Is Required for SUMO Processing but Not for Viability.

The protozoan parasite Leishmania donovani is part of an early eukaryotic branch and depends on post-transcriptional mechanisms for gene expression regulation. This includes post-transcriptional protein modifications, such as protein phosphorylation. The presence of genes for protein SUMOylation, i.e., the covalent attachment of small ubiquitin-like modifier (SUMO) polypeptides, in the Leishmania genomes prompted us to investigate the importance of the sentrin-specific protease (SENP) and its putative client, SUMO, for the vitality and infectivity of Leishmania donovani. While SENP null mutants are viable with reduced vitality, viable SUMO null mutant lines could not be obtained. SUMO C-terminal processing is disrupted in SENP null mutants, preventing SUMO from covalent attachment to proteins and nuclear translocation. Infectivity in vitro is not affected by the loss of SENP-dependent SUMO processing. We conclude that SENP is required for SUMO processing, but that functions of unprocessed SUMO are critical for Leishmania viability.

Human CD8+ T Cells Release Extracellular Traps Co-Localized With Cytotoxic Vesicles That Are Associated With Lesion Progression and Severity in Human Leishmaniasis.

Cell death plays a fundamental role in mounting protective and pathogenic immunity. Etosis is a cell death mechanism defined by the release of extracellular traps (ETs), which can foster inflammation and exert microbicidal activity. While etosis is often associated with innate cells, recent studies showed that B cells and CD4+ T cells can release ETs. Here we investigate whether CD8+ T cells can also release ETs, which might be related to cytotoxicity and tissue pathology. To these ends, we first employed an in vitro system stimulating human CD8+ T cells isolated from healthy volunteers with anti-CD3/anti-CD28. Using time-frame video, confocal and electron microscopy, we demonstrate that human CD8+ T cells release ETs upon stimulation (herein LETs - lymphocyte extracellular traps), which display unique morphology and functional characteristics. CD8+ T cell-derived LETs form long strands that co-localize with CD107a, a marker of vesicles containing cytotoxic granules. In addition, these structures connect the LET-releasing cell to other neighboring cells, often resulting in cell death. After demonstrating the release of LETs by human CD8+ T cells in vitro, we went on to study the occurrence of CD8-derived LETs in a human disease setting. Thus, we evaluated the occurrence of CD8-derived LETs in lesions from patients with human tegumentary leishmaniasis, where CD8+ T cells play a key role in mediating pathology. In addition, we evaluated the association of these structures with the intensity of the inflammatory infiltrate in early and late cutaneous, as well as in mucosal leishmaniasis lesions. We demonstrated that progression and severity of debilitating and mutilating forms of human tegumentary leishmaniasis are associated with the frequency of CD8+ T cells in etosis, as well as the occurrence of CD8-derived LETs carrying CD107a+ vesicles in the lesions. We propose that CD8+ T cell derived LETs may serve as a tool for delivering cytotoxic vesicles to distant target cells, providing insights into mechanisms of CD8+ T cell mediated pathology.