Leishmania (Leishmania) amazonensis is an intracellular protozoan parasite responsible for the cutaneous leishmaniasis. The parasite replicates inside mammalian macrophage to establish infection. Host-pathogen interactions result in microRNA-mediated post-transcriptional regulation of host genes involved in inflammatory immune response. We analyzed macrophage miRNA profiles during L. (L.) amazonensis infection. The regulation of macrophage miRNA expression by the parasite correlates with/depends on parasite arginase activity during infection. L. (L.) amazonensis (La-WT) presented significant miRNA profile alteration (27%) compared to L. (L.) amazonensis arginase knockout (La-arg−) (~40%) in relation to uninfected-macrophages. We observed that 78% of the altered miRNAs were up-regulated in La-WT infection, while only 32% were up-regulated in La-arg−-infected macrophages. In contrast to La-WT, the lack of L. (L.) amazonensis arginase led to the inhibition of miR-294 and miR-721 expression. The expression of miR-294 and miR-721 was recovered to levels similar to La-WT in La-arg− addback mutant. The inhibition of miR-294/Nos2 and miR721/Nos2 interactions increased NOS2 expression and NO production, and reduced L. (L.) amazonensis infectivity, confirming Nos2 as target of these miRNAs. The role of miR-294 and miR-721 in the regulation of NOS2 expression during Leishmania replication in infected macrophages pointing these miRNAs as potential new targets for drug development.

Leishmania is a protozoan parasite that alternates its life cycle between the sand fly and the mammalian host macrophages, involving several environmental changes. The parasite responds to these changes by promoting a rapid metabolic adaptation through cellular signaling modifications that lead to transcriptional and post-transcriptional gene expression regulation and morphological modifications. Molecular approaches such as gene expression regulation, next-generation sequencing (NGS), microRNA (miRNA) expression profiling, in cell Western blot analyses and enzymatic activity profiling, have been used to characterize the infection of murine BALB/c and C57BL/6 macrophages, as well as the human monocytic cell-lineage THP-1, with Leishmania amazonensis wild type (La-WT) or arginase knockout (La-arg-). These models are being used to elucidate physiological roles of arginine and polyamines pathways and the importance of arginase for the establishment of the infection. In this review, we will describe the main aspects of Leishmania-host interaction, focusing on the arginine and polyamines pathways and pointing to possible targets to be used for prognosis and/or in the control of the infection. The parasite enzymes, arginase and nitric oxide synthase-like, have essential roles in the parasite survival and in the maintenance of infection. On the other hand, in mammalian macrophages, defense mechanisms are activated inducing alterations in the mRNA, miRNA and enzymatic profiles that lead to the control of infection. Furthermore, the genetic background of both parasite and host are also important to define the fate of infection.

Iron, an essential co-factor of respiratory chain proteins, is critical for mitochondrial function and maintenance of its redox balance. We previously reported a role for iron uptake in differentiation of Leishmania amazonensis into virulent amastigotes, by a mechanism that involves reactive oxygen species (ROS) production and is independent of the classical pH and temperature cues. Iron import into mitochondria was proposed to be essential for this process, but evidence supporting this hypothesis was lacking because the Leishmania mitochondrial iron transporter was unknown. Here we describe MIT1, a homolog of the mitochondrial iron importer genes mrs3 (yeast) and mitoferrin-1 (human) that is highly conserved among trypanosomatids. MIT1 expression was essential for the survival of Trypanosoma brucei procyclic but not bloodstream forms, which lack functional respiratory complexes. L. amazonensis LMIT1 null mutants could not be generated, suggesting that this mitochondrial iron importer is essential for promastigote viability. Promastigotes lacking one LMIT1 allele (LMIT1/Δlmit1) showed growth defects and were more susceptible to ROS toxicity, consistent with the role of iron as the essential co-factor of trypanosomatid mitochondrial superoxide dismutases. LMIT1/Δlmit1 metacyclic promastigotes were unable to replicate as intracellular amastigotes after infecting macrophages or cause cutaneous lesions in mice. When induced to differentiate axenically into amastigotes, LMIT1/Δlmit1 showed strong defects in iron content and function of mitochondria, were unable to upregulate the ROS-regulatory enzyme FeSOD, and showed mitochondrial changes suggestive of redox imbalance. Our results demonstrate the importance of mitochondrial iron uptake in trypanosomatid parasites, and highlight the role of LMIT1 in the iron-regulated process that orchestrates differentiation of L. amazonensis into infective amastigotes.

Acute inflammatory responses induced by bacteria or fungi block nocturnal melatonin synthesis by rodent pineal glands. Here, we show Leishmania infection does not impair daily melatonin rhythm in hamsters. Remarkably, the attenuated parasite burden and lesion progression in hamsters infected at nighttime was impaired by blockage of melatonin receptors with luzindole, whereas melatonin treatment during the light phase attenuated Leishmania infection. In vitro studies corroborated in vivo observations. Melatonin treatment reduced macrophage expression of Cat-2b, Cat1, and ArgI, genes involved in arginine uptake and polyamine synthesis. Indeed, melatonin reduced macrophage arginine uptake by 40%. Putrescine supplementation reverted the attenuation of infectivity by melatonin indicating that its effect was due to the arrest of parasite replication. This study shows that the Leishmania/host interaction varies in a circadian manner according to nocturnal melatonin pineal synthesis. Our results provide new data regarding Leishmania infectiveness and show new approaches for applying agonists of melatonin receptors in Leishmaniasis therapy.

Lipopolysaccharide (LPS) modulates the transcription of the gene that codifies the enzyme arylalkylamine-N-acetyltransferase (AA-NAT) through nuclear translocation of the transcription factor nuclear factor-κ-light-chain-enhancer of activated B cells (NF-κB). AA-NAT converts serotonin to N-acetylserotonin, the ultimate precursor of melatonin. Activation of kappa B elements (aa-nat-κB), localized in the promoter (nat-κB1 and nat-κB2), leads to Aa-nat transcription in RAW 264.7 macrophages. Competitive electrophoretic mobility shift assay (EMSA) with oligonucleotide probes corresponding to each of the two elements, as well as a NF-κB consensus corresponding probe, revealed different specificities for each κB element. In addition, activator protein-1 (AP-1) as well as signal transducers and activator of transcription-1 and 3 (STAT-1; STAT-3) competed with NF-κB for binding to nat-κB1, while only STAT-3 competed with NF-κB for binding to nat-κB2. According to co-immunoprecipitation (ChiP) assays, these two sites are able to distinguish NF-κB subunits. The sequence nat-κB1 bound dimers containing p52, RelA, and cRel, while nat-κB2 bound preferentially p50, p52, and RelA, and did not bind cRel. The expression of RelA and cRel is essential for the induction of Aa-nat expression and melatonin synthesis. Considering that the expression of cRel is induced by the earlier expressed p50/RelA, the differential effects of NF-κB dimers may be intimately associated with the temporal regulation of inflammatory responses, with the resolution phase being associated with paracrine and autocrine melatonin effects. Such data suggest that the proven effects of exogenous melatonin in the resolution phase of inflammation are paralleled by the effects of locally synthesized melatonin in immune cells.