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.

STAT1-NFκB crosstalk triggered by interferon gamma regulates noradrenaline-induced pineal hormonal production.

Melatonin production by pineal glands is modulated by several immune signals. The nuclear translocation of nuclear factor kappa-B (NFκB) homodimers, lacking transactivation domains, once induced by lipopolysaccharide (LPS) or tumor necrosis factor (TNF), inhibits the expression of Aanat gene and the synthesis of noradrenaline (NA)-induced melatonin. Interferon gamma (IFN-γ), on the other hand, increases melatonin synthesis. Furthermore, this cytokine activates the signal transducer as well as the activator of transcription 1 (STAT1) pathway, which was never evaluated as a melatonin synthesis modulator before. Reports demonstrated that IFN-γ might also activate NFκB. The present study evaluated the role of STAT1-NFκB crosstalk triggered by IFN-γ regarding the regulation of NA-induced pineal glands' hormonal production. Moreover, IFN-γ treatment increased NA-induced Aanat transcription, in addition to the synthesis of N-acetylserotonin (NAS) and melatonin. These effects were associated with STAT1 nuclear translocation, confirmed by the co-immunoprecipitation of STAT1 and Aanat promoter. Pharmacological STAT1 enhancement augmented NA-induced Aanat transcription as well as NAS and melatonin production. Additionally, IFN-γ induced the nuclear translocation of RelA-NFκB subunits. The blockade of this pathway prevented IFN-γ effects on the pineal function. The present data show that STAT1 and NFκB crosstalk controls melatonin production through a synergistic mechanism, disclosing a new integrative mechanism regarding pineal hormonal activity control.

Metabolomic Profile of BALB/c Macrophages Infected with Leishmania amazonensis: Deciphering L-Arginine Metabolism.

BACKGROUND: Leishmaniases are neglected tropical diseases that are caused by Leishmania, being endemic worldwide. L-arginine is an essential amino acid that is required for polyamines production on mammal cells. During Leishmania infection of macrophages, L-arginine is used by host and parasite arginase to produce polyamines, leading to parasite survival; or, by nitric oxide synthase 2 to produce nitric oxide leading to parasite killing. Here, we determined the metabolomic profile of BALB/c macrophages that were infected with L. amazonensis wild type or with L. amazonensis arginase knockout, correlating the regulation of L-arginine metabolism from both host and parasite. METHODS: The metabolites of infected macrophages were analyzed by capillary electrophoresis coupled with mass spectrometry (CE-MS). The metabolic fingerprints analysis provided the dual profile from the host and parasite. RESULTS: We observed increased levels of proline, glutamic acid, glutamine, L-arginine, ornithine, and putrescine in infected-L. amazonensis wild type macrophages, which indicated that this infection induces the polyamine production. Despite this, we observed reduced levels of ornithine, proline, and trypanothione in infected-L. amazonensis arginase knockout macrophages, indicating that this infection reduces the polyamine production. CONCLUSIONS: The metabolome fingerprint indicated that Leishmania infection alters the L-arginine/polyamines/trypanothione metabolism inside the host cell and the parasite arginase impacts on L-arginine metabolism and polyamine production, defining the infection fate.

The RelA/cRel nuclear factor-κB (NF-κB) dimer, crucial for inflammation resolution, mediates the transcription of the key enzyme in melatonin synthesis in RAW 264.7 macrophages.

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.

Capillary electrophoresis reveals polyamine metabolism modulation in Leishmania (Leishmania) amazonensis wild-type and arginase-knockout mutants under arginine starvation.

l-Arginine is an essential amino acid in Leishmania (Leishmania) amazonensis metabolism. A key enzyme for parasite l-arginine metabolism is arginase (ARG) that uses arginine to produce urea and ornithine, a precursor of polyamine pathway guaranteeing parasite replication in both insect and mammal hosts. There is an alternative pathway to produce ornithine via l-proline and glutamate, but this mechanism is not described in Leishmania. In the mammal host, two enzymes can use l-arginine as substrate, the host ARG and the induced nitric oxide synthase that produces nitric oxide. The competition between induced nitric oxide synthase and both parasite and host ARG can favor the success of the infection or its control. Here, we established the metabolomics profile of the polyamine pathway of wild type (WT) L. (L.) amazonensis, submitted or not to l-arginine starvation, and compared to the ARG-knockout mutant (arg- ). Our results indicated that arginine starvation induces a decrease in arginine, ornithine, and putrescine, but we could not detect the significative level changes of spermidine, spermine, or agmatine. However, the absence of ARG on the arg- induced an increase of arginine and citrulline levels, but decreased the levels of ornithine and putrescine. Similarly to the WT arginine-starved parasites, the arg- parasites presented lower levels of proline when compared to the WT ones. This could be indicative of an alternative pathway to surpass the enzyme or its substrate absence.

Blocking the CTLA-4 and PD-1 pathways during pulmonary paracoccidioidomycosis improves immunity, reduces disease severity, and increases the survival of infected mice.

Immune checkpoint pathways, i.e., coinhibitory pathways expressed as feedback following immune activation, are crucial for controlling an excessive immune response. Cytotoxic T lymphocyte antigen-4 (CTLA-4) and programmed cell death protein-1 (PD-1) are the central classical checkpoint inhibitory (CPI) molecules used for the control of neoplasms and some infectious diseases, including some fungal infections. As the immunosuppression of severe paracoccidioidomycosis (PCM), a chronic granulomatous fungal disease, was shown to be associated with the expression of coinhibitory molecules, we hypothesized that the inhibition of CTLA-4 and PD-1 could have a beneficial effect on pulmonary PCM. To this end, C57BL/6 mice were infected with Paracoccidioides brasiliensis yeasts and treated with monoclonal antibodies (mAbs) α-CTLA-4, α-PD-1, control IgG, or PBS. We verified that blockade of CTLA-4 and PD-1 reduced the fungal load in the lungs and fungal dissemination to the liver and spleen and decreased the size of pulmonary lesions, resulting in increased survival of mice. Compared with PBS-treated infected mice, significantly increased levels of many pro- and anti-inflammatory cytokines were observed in the lungs of α-CTLA-4-treated mice, but a drastic reduction in the liver was observed following PD-1 blockade. In the lungs of α-CPI and IgG-treated mice, there were no changes in the frequency of inflammatory leukocytes, but a significant reduction in the total number of these cells was observed. Compared with PBS-treated controls, α-CPI- and IgG-treated mice exhibited reduced pulmonary infiltration of several myeloid cell subpopulations and decreased expression of costimulatory molecules. In addition, a decreased number of CD4+ and CD8+ T cells but sustained numbers of Th1, Th2, and Th17 T cells were detected. An expressive reduction in several Treg subpopulations and their maturation and suppressive molecules, in addition to reduced numbers of Treg, TCD4+, and TCD8+ cells expressing costimulatory and coinhibitory molecules of immunity, were also detected. The novel cellular and humoral profiles established in the lungs of α-CTLA-4 and α-PD-1-treated mice but not in control IgG-treated mice were more efficient at controlling fungal growth and dissemination without causing increased tissue pathology due to excessive inflammation. This is the first study demonstrating the efficacy of CPI blockade in the treatment of pulmonary PCM, and further studies combining the use of immunotherapy with antifungal drugs are encouraged.

Putrescine supplementation shifts macrophage L-arginine metabolism related-genes reducing Leishmania amazonensis infection.

Leishmania is a protozoan that causes leishmaniasis, a neglected tropical disease with clinical manifestations classified as cutaneous, mucocutaneous, and visceral leishmaniasis. In the infection context, the parasite can modulate macrophage gene expression affecting the microbicidal activity and immune response. The metabolism of L-arginine into polyamines putrescine, spermidine, and spermine reduces nitric oxide (NO) production, favoring Leishmania survival. Here, we investigate the effect of supplementation with L-arginine and polyamines in infection of murine BALB/c macrophages by L. amazonensis and in the transcriptional regulation of genes involved in arginine metabolism and proinflammatory response. We showed a reduction in the percentage of infected macrophages upon putrescine supplementation compared to L-arginine, spermidine, and spermine supplementation. Unexpectedly, deprivation of L-arginine increased nitric oxide synthase (Nos2) gene expression without changes in NO production. Putrescine supplementation increased transcript levels of polyamine metabolism-related genes Arg2, ornithine decarboxylase (Odc1), Spermidine synthase (SpdS), and Spermine synthase (SpmS), but reduced Arg1 in L. amazonensis infected macrophages, while spermidine and spermine promoted opposite effects. Putrescine increased Nos2 expression without leading to NO production, while L-arginine plus spermine led to NO production in uninfected macrophages, suggesting that polyamines can induce NO production. Besides, L-arginine supplementation reduced Il-1b during infection, and L-arginine or L-arginine plus putrescine increased Mcp1 at 24h of infection, suggesting that polyamines availability can interfere with cytokine/chemokine production. Our data showed that putrescine shifts L-arginine-metabolism related-genes on BALB/c macrophages and affects infection by L. amazonensis.

miR-294 and miR-410 Negatively Regulate Tnfa, Arginine Transporter Cat1/2, and Nos2 mRNAs in Murine Macrophages Infected with Leishmania amazonensis.

MicroRNAs are small non-coding RNAs that regulate cellular processes by the post-transcriptional regulation of gene expression, including immune responses. The shift in the miRNA profiling of murine macrophages infected with Leishmania amazonensis can change inflammatory response and metabolism. L-arginine availability and its conversion into nitric oxide by nitric oxide synthase 2 (Nos2) or ornithine (a polyamine precursor) by arginase 1/2 regulate macrophage microbicidal activity. This work aimed to evaluate the function of miR-294, miR-301b, and miR-410 during early C57BL/6 bone marrow-derived macrophage infection with L. amazonensis. We observed an upregulation of miR-294 and miR-410 at 4 h of infection, but the levels of miR-301b were not modified. This profile was not observed in LPS-stimulated macrophages. We also observed decreased levels of those miRNAs target genes during infection, such as Cationic amino acid transporters 1 (Cat1/Slc7a1), Cat2/Slc7a22 and Nos2; genes were upregulated in LPS stimuli. The functional inhibition of miR-294 led to the upregulation of Cat2 and Tnfa and the dysregulation of Nos2, while miR-410 increased Cat1 levels. miR-294 inhibition reduced the number of amastigotes per infected macrophage, showing a reduction in the parasite growth inside the macrophage. These data identified miR-294 and miR-410 biomarkers for a potential regulator in the inflammatory profiles of microphages mediated by L. amazonensis infection. This research provides novel insights into immune dysfunction contributing to infection outcomes and suggests the use of the antagomiRs/inhibitors of miR-294 and miR-410 as new therapeutic strategies to modulate inflammation and to decrease parasitism.

Proteome and morphological analysis show unexpected differences between promastigotes of Leishmania amazonensis PH8 and LV79 strains.

BACKGROUND: Leishmaniases are diseases caused by Leishmania protozoans that affect around 12 million people. Leishmania promastigotes are transmitted to vertebrates by female phlebotomine flies during their blood meal. Parasites attach to phagocytic cells, are phagocytosed and differentiate into amastigotes. We previously showed that PH8 and LV79 strains of Leishmania amazonensis have different virulence in mice and that their amastigotes differ in their proteomes. In this work, we compare promastigotes' infectivity in macrophages, their proteomes and morphologies. METHODS/PRINCIPAL FINDINGS: Phagocytosis assays showed that promastigotes adhesion to and phagocytosis by macrophages is higher in PH8 than LV79. To identify proteins that differ between the two strains and that may eventually contribute for these differences we used a label-free proteomic approach to compare promastigote´s membrane-enriched fractions. Proteomic analysis enabled precise discrimination of PH8 and LV79 protein profiles and the identification of several differentially abundant proteins. The proteins more abundant in LV79 promastigotes participate mainly in translation and amino acid and nucleotide metabolism, while the more abundant in PH8 are involved in carbohydrate metabolism, cytoskeleton composition and vesicle/membrane trafficking. Interestingly, although the virulence factor GP63 was more abundant in the less virulent LV79 strain, zymography suggests a higher protease activity in PH8. Enolase, which may be related to virulence, was more abundant in PH8 promastigotes. Unexpectedly, flow cytometry and morphometric analysis indicate higher abundance of metacyclics in LV79. CONCLUSIONS/SIGNIFICANCE: Proteome comparison of PH8 and LV79 promastigotes generated a list of differential proteins, some of which may be further prospected to affect the infectivity of promastigotes. Although proteomic profile of PH8 includes more proteins characteristic of metacyclics, flow cytometry and morphometric analysis indicate a higher abundance of metacyclics in LV79 cultures. These results shed light to the gaps in our knowledge of metacyclogenesis in L. amazonensis, and to proteins that should be studied in the context of infection by this species.

Corrigendum: miR-548d-3p Is Up-regulated in Human Visceral Leishmaniasis and Suppresses Parasite Growth in Macrophages.

[This corrects the article DOI: 10.3389/fcimb.2022.826039.].

miR-548d-3p Is Up-Regulated in Human Visceral Leishmaniasis and Suppresses Parasite Growth in Macrophages.

Visceral leishmaniasis caused by Leishmania (Leishmania) infantum in Latin America progress with hepatosplenomegaly, pancytopenia, hypergammaglobulinemia, and weight loss and maybe lethal mainly in untreated cases. miRNAs are important regulators of immune and inflammatory gene expression, but their mechanisms of action and their relationship to pathogenesis in leishmaniasis are not well understood. In the present study, we sought to quantify changes in miRNAs associated with immune and inflammatory pathways using the L. (L.) infantum promastigote infected- human monocytic THP-1 cell model and plasma from patients with visceral leishmaniasis. We identified differentially expressed miRNAs in infected THP-1 cells compared with non-infected cells using qPCR arrays. These miRNAs were submitted to in silico analysis, revealing targets within functional pathways associated with TGF-ß, chemokines, glucose metabolism, inflammation, apoptosis, and cell signaling. In parallel, we identified differentially expressed miRNAs in active visceral leishmaniasis patient plasma compared with endemic healthy controls. In silico analysis of these data indicated different predicted targets within the TGF-ß, TLR4, IGF-I, chemokine, and HIF1α pathways. Only a small number of miRNAs were commonly identified in these two datasets, notably with miR-548d-3p being up-regulated in both conditions. To evaluate the potential biological role of miR-548d-3p, we transiently transfected a miR-548d-3p inhibitor into L. (L.) infantum infected-THP-1 cells, finding that inhibition of miR-548d-3p enhanced parasite growth, likely mediated through reduced levels of MCP-1/CCL2 and nitric oxide production. Further work will be required to determine how miR-548d-3p plays a role in vivo and whether it serves as a potential biomarker of progressive leishmaniasis.

miR-548d-3p Alters Parasite Growth and Inflammation in Leishmania (Viannia) braziliensis Infection.

American Tegumentary Leishmaniasis (ATL) is an endemic disease in Latin America, mainly caused in Brazil by Leishmania (Viannia) braziliensis. Clinical manifestations vary from mild, localized cutaneous leishmaniasis (CL) to aggressive mucosal disease. The host immune response strongly determines the outcome of infection and pattern of disease. However, the pathogenesis of ATL is not well understood, and host microRNAs (miRNAs) may have a role in this context. In the present study, miRNAs were quantified using qPCR arrays in human monocytic THP-1 cells infected in vitro with L. (V.) braziliensis promastigotes and in plasma from patients with ATL, focusing on inflammatory response-specific miRNAs. Patients with active or self-healed cutaneous leishmaniasis patients, with confirmed parasitological or immunological diagnosis, were compared with healthy controls. Computational target prediction of significantly-altered miRNAs from in vitro L. (V.) braziliensis-infected THP-1 cells revealed predicted targets involved in diverse pathways, including chemokine signaling, inflammatory, cellular proliferation, and tissue repair processes. In plasma, we observed distinct miRNA expression in patients with self-healed and active lesions compared with healthy controls. Some miRNAs dysregulated during THP-1 in vitro infection were also found in plasma from self-healed patients, including miR-548d-3p, which was upregulated in infected THP-1 cells and in plasma from self-healed patients. As miR-548d-3p was predicted to target the chemokine pathway and inflammation is a central to the pathogenesis of ATL, we evaluated the effect of transient transfection of a miR-548d-3p inhibitor on L. (V.) braziliensis infected-THP-1 cells. Inhibition of miR-548d-3p reduced parasite growth early after infection and increased production of MCP1/CCL2, RANTES/CCL5, and IP10/CXCL10. In plasma of self-healed patients, MCP1/CCL2, RANTES/CCL5, and IL-8/CXCL8 concentrations were significantly decreased and MIG/CXCL9 and IP-10/CXCL10 increased compared to patients with active disease. These data suggest that by modulating miRNAs, L. (V.) braziliensis may interfere with chemokine production and hence the inflammatory processes underpinning lesion resolution. Our data suggest miR-548d-3p could be further evaluated as a prognostic marker for ATL and/or as a host-directed therapeutic target.

Atypical Prolonged Viral Shedding With Intra-Host SARS-CoV-2 Evolution in a Mildly Affected Symptomatic Patient.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is caused by a respiratory virus with a wide range of manifestations, varying from asymptomatic to fatal cases, with a generally short outcome. However, some individuals present long-term viral shedding. We monitored 38 individuals who were mildly affected by the SARS-CoV-2 infection. Out of the total studied population, three (7.9%) showed atypical events regarding the duration of positivity for viral RNA detection. In one of these atypical cases, a previously HIV-positive male patient presented a SARS-CoV-2 RNA shedding and subgenomic RNA (sgRNA) detected from the upper respiratory tract, respectively, for 232 and 224 days after the onset of the symptoms. The SARS-CoV-2 B.1.1.28 lineage, one of the most prevalent in Brazil in 2020, was identified in this patient in three serial samples. Interestingly, the genomic analyses performed throughout the infectious process showed an increase in the genetic diversity of the B.1.1.28 lineage within the host itself, with viral clearance occurring naturally, without any intervention measures to control the infection. Contrasting widely spread current knowledge, our results indicate that potentially infectious SARS-CoV-2 virus might be shed by much longer periods by some infected patients. This data call attention to better adapted non-pharmacological measures and clinical discharge of patients aiming at preventing the spread of SARS-CoV-2 to the population.

Gradual decline in malaria-specific memory T cell responses leads to failure to maintain long-term protective immunity to Plasmodium chabaudi AS despite persistence of B cell memory and circulating antibody.

The mechanisms responsible for the generation and maintenance of immunological memory to Plasmodium are poorly understood and the reasons why protective immunity in humans is so difficult to achieve and rapidly lost remain a matter for debate. A possible explanation for the difficulty in building up an efficient immune response against this parasite is the massive T cell apoptosis resulting from exposure to high-dose parasite Ag. To determine the immunological mechanisms required for long-term protection against P. chabaudi malaria and the consequences of high and low acute phase parasite loads for acquisition of protective immunity, we performed a detailed analysis of T and B cell compartments over a period of 200 days following untreated and drug-treated infections in female C57BL/6 mice. By comparing several immunological parameters with the capacity to control a secondary parasite challenge, we concluded that loss of full protective immunity is not determined by acute phase parasite load nor by serum levels of specific IgG2a and IgG1 Abs, but appears to be a consequence of the progressive decline in memory T cell response to parasites, which occurs similarly in untreated and drug-treated mice with time after infection. Furthermore, by analyzing adoptive transfer experiments, we confirmed the major role of CD4(+) T cells for guaranteeing long-term full protection against P. chabaudi malaria.

MicroRNAs: Biological Regulators in Pathogen-Host Interactions.

An inflammatory response is essential for combating invading pathogens. Several effector components, as well as immune cell populations, are involved in mounting an immune response, thereby destroying pathogenic organisms such as bacteria, fungi, viruses, and parasites. In the past decade, microRNAs (miRNAs), a group of noncoding small RNAs, have emerged as functionally significant regulatory molecules with the significant capability of fine-tuning biological processes. The important role of miRNAs in inflammation and immune responses is highlighted by studies in which the regulation of miRNAs in the host was shown to be related to infectious diseases and associated with the eradication or susceptibility of the infection. Here, we review the biological aspects of microRNAs, focusing on their roles as regulators of gene expression during pathogen-host interactions and their implications in the immune response against Leishmania, Trypanosoma, Toxoplasma, and Plasmodium infectious diseases.

Author Correction: Differential immune response modulation in early Leishmania amazonensis infection of BALB/c and C57BL/6 macrophages based on transcriptome profiles.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Dual transcriptome analysis reveals differential gene expression modulation influenced by Leishmania arginase and host genetic background.

The outcome of Leishmania infection is strongly influenced by the host's genetic background. BALB/c mice are susceptible to Leishmania infection, while C57BL/6 mice show discrete resistance. Central to the fate of the infection is the availability of l-arginine and the related metabolic processes in the host and parasite. Depending on l-arginine availability, nitric oxide synthase 2 (NOS2) of the host cell produces nitric oxide (NO) controlling the parasite growth. On the other hand, Leishmania can also use host l-arginine for the production of polyamines through its own arginase activity, thus favouring parasite replication. Considering RNA-seq data, we analysed the dual modulation of host and parasite gene expression of BALB/c or C57BL/6 mouse bone marrow-derived macrophages (BMDMs) after 4 h of infection with Leishmania amazonensis wild-type (La-WT) or L. amazonensis arginase knockout (La-arg-). We identified 12 641 host transcripts and 8282 parasite transcripts by alignment analysis with the respective Mus musculus and L. mexicana genomes. The comparison of BALB/c_La-arg-versus BALB/c_La-WT revealed 233 modulated transcripts, with most related to the immune response and some related to the amino acid transporters and l-arginine metabolism. In contrast, the comparison of C57BL/6_La-arg-vs. C57BL/6_La-WT revealed only 30 modulated transcripts, including some related to the immune response but none related to amino acid transport or l-arginine metabolism. The transcriptome profiles of the intracellular amastigote revealed 94 modulated transcripts in the comparison of La-arg-_BALB/c vs. La-WT_BALB/c and 45 modulated transcripts in the comparison of La-arg-_C57BL/6 vs. La-WT_C57BL/6. Taken together, our data present new insights into the impact of parasite arginase activity on the orchestration of the host gene expression modulation, including in the immune response and amino acid transport and metabolism, mainly in susceptible BALB/c-infected macrophages. Moreover, we show how parasite arginase activity affects parasite gene expression modulation, including amino acid uptake and amastin expression.

Differential expression of polyamine biosynthetic pathways in skin lesions and in plasma reveals distinct profiles in diffuse cutaneous leishmaniasis.

Tegumentary leishmaniasis (TL) is a parasitic disease that can result in wide spectrum clinical manifestations. It is necessary to understand host and parasite determinants of clinical outcomes to identify novel therapeutic targets. Previous studies have indicated that the polyamine biosynthetic pathway is critical for Leishmania growth and survival. Despite its importance, expression of the such pathway has not been previously investigated in TL patients. We performed an exploratory analysis employing Systems Biology tools to compare circulating polyamines and amino acid concentration as well as polyamine pathway gene expression in cutaneous lesions patients presenting with distinct TL disease presentations. Diffuse cutaneous leishmaniasis (DCL) was associated with higher concentrations of amino acids, polyamines and its substrate transporters than mucosal cutaneous leishmaniasis or localized cutaneous leishmaniasis. In addition, the RNA expression of polyamine-related genes of patients lesions from two separate cohorts demonstrated that differential activation of this pathway is associated with parasite loads and able to discriminate the clinical spectrum of TL. Taken together, our findings highlight a new aspect of DCL immunopathogenesis indicating that the polyamine pathway may be explored as a novel therapeutic target to control disease burden.

The impact of arginase activity on virulence factors of Leishmania amazonensis.

The outcome of Leishmania infection depends on the parasite species and the host immune response. Virulence factors have been extensively studied over the years in an effort to find efficient vaccines and/or treatments for Leishmania infection. Arginase activity in Leishmania has been described as an essential player for the polyamines pathway, impacting parasite replication and infectivity. Considering previous studies showing that the absence of arginase activity leads to low infectivity of Leishmania amazonensis, we reanalyzed transcriptomic data comparing both promastigotes and axenic amastigotes from L. amazonensis wild type (La-WT) and L. amazonensis arginase knockout (La-arg-) backgrounds. The analysis produced a new compilation of modulated transcripts that indicated the role of arginase not only in the polyamines pathway but also in the modulation of virulence factors involved in parasite recognition, growth and differentiation.

Differential immune response modulation in early Leishmania amazonensis infection of BALB/c and C57BL/6 macrophages based on transcriptome profiles.

The fate of Leishmania infection can be strongly influenced by the host genetic background. In this work, we describe gene expression modulation of the immune system based on dual global transcriptome profiles of bone marrow-derived macrophages (BMDMs) from BALB/c and C57BL/6 mice infected with Leishmania amazonensis. A total of 12,641 host transcripts were identified according to the alignment to the Mus musculus genome. Differentially expressed genes (DEGs) profiling revealed a differential modulation of the basal genetic background between the two hosts independent of L. amazonensis infection. In addition, in response to early L. amazonensis infection, 10 genes were modulated in infected BALB/c vs. non-infected BALB/c macrophages; and 127 genes were modulated in infected C57BL/6 vs. non-infected C57BL/6 macrophages. These modulated genes appeared to be related to the main immune response processes, such as recognition, antigen presentation, costimulation and proliferation. The distinct gene expression was correlated with the susceptibility and resistance to infection of each host. Furthermore, upon comparing the DEGs in BMDMs vs. peritoneal macrophages, we observed no differences in the gene expression patterns of Jun, Fcgr1 and Il1b, suggesting a similar activation trends of transcription factor binding, recognition and phagocytosis, as well as the proinflammatory cytokine production in response to early L. amazonensis infection. Analysis of the DEG profile of the parasite revealed only one DEG among the 8,282 transcripts, indicating that parasite gene expression in early infection does not depend on the host genetic background.