Polyamines regulate the expression of genes related to its metabolism and resistance to infection of macrophages with Leishmania amazonensis

Os macrófagos são células fagocíticas do sistema imune e representam a primeira barreira frente à infecção com Leishmania. Leishmania é um protozoário causador das leishmanioses, doenças que apresentam manifestações clínicas classificadas em leishmaniose cutânea, mucocutânea e visceral. No contexto da infecção, o parasita é capaz de modular a expressão de genes do macrófago que afetam a atividade microbicida e a resposta imune frente ao parasita. A metabolização de L-arginina em poliaminas (putrescina, espermidina e espermina) em detrimento da produção de óxido nítrico (NO) favorece a infecção com Leishmania. As poliaminas são moléculas essenciais para a síntese proteica e síntese de macromoléculas nas células, e também proliferação e diferenciação celular. No presente estudo, nós avaliamos como a disponibilidade de L-arginina e poliaminas afeta a expressão de genes relacionados ao seu metabolismo em macrófagos de camundongos BALB/c infectados com Leishmania amazonensis e susceptibilidade à infecção. A privação de L-arginina em macrófagos durante a infecção com L. amazonensis aumentou a expressão de Nos2, mas não induziu produção de NO. Contudo, a suplementação com L-arginina não alterou a expressão de genes relacionados ao seu transporte e produção de poliaminas. A suplementação com L-arginina e putrescina aumentou a expressão de arginase 2 (Arg2) em macrófagos infectados comparado aos não infectados, enquanto a suplementação com espermidina ou espermina reduziu a expressão de Arg2. A suplementação com L-arginina e espermina aumentou a expressão de espermidina sintase (SpdS) em macrófagos infectados comparado aos não infectados. A suplementação com putrescina aumentou a expressão de Nos2, mas não aumentou a produção de NO durante a infecção. Enquanto, a suplementação dos macrófagos com espermina durante a infecção aumentou a frequência de células produtoras de NO. Os genes Slc3a2/Slc7a5 que compõem o heterodímero do transportador de poliaminas foram modulados. Houve o aumento na expressão de Slc3a2 em macrófagos suplementados com putrescina ou L-arginina e espermidina e de Slc7a5 na suplementação com espermidina ou espermina durante a infecção. A suplementação com putrescina também aumentou a expressão do gene da proteína quimioatraente de monócito (Mcp1) após a infecção. A análise de infectividade mostrou que a suplementação com putrescina, L-arginina e espermidina ou L-arginina e espermina reduziu a porcentagem de macrófagos infectados quando comparamos com a suplementação com L-arginina. Nossos dados sugerem que as poliaminas regulam a expressão de genes relacionados ao seu transporte e metabolismo em macrófagos e afetam a infecção com L. amazonensis.Macrophages are phagocytic cells of the immune system and represent the first barrier against infection with Leishmania. Leishmania is a protozoan that causes leishmaniasis, a disease that presents clinical manifestations classified as cutaneous, mucocutaneous and visceral leishmaniasis. In the infection context, the parasite can modulate macrophage gene expression affecting microbicidal activity and immune response to the parasite. The metabolization of L-arginine in polyamines (putrescine, spermidine, and spermine) harmful to the nitric oxide (NO) production favoring Leishmania infection. Polyamines are essential for protein and macromolecules synthesis in cells, and cell proliferation and differentiation. In the present study, we evaluated how the availability of L-arginine and polyamines affected the expression of genes related to their metabolism in macrophages of BALB/c mice infected with Leishmania amazonensis and susceptibility to infection. L-arginine deprivation in macrophages during infection with L. amazonensis increased Nos2 expression, but did not induce NO production. However, L-arginine supplementation did not alter the expression of genes related to polyamines transport and biosynthesis. Supplementation with L-arginine and putrescine increased the expression of arginase 2 (Arg2) in infected-macrophages in relation to uninfected macrophages, while the supplementation with spermidine or spermine reduced the expression of Arg2. Supplementation with L-arginine and spermine increased the expression of spermidine synthase (SpdS) in infected macrophages compared to uninfected ones. Putrescine supplementation increased Nos2 expression, but did not increase NO production during infection. Meanwhile, the spermine supplementation of macrophages during infection increased the frequency of NO-producing cells. The Slc3a2/Slc7a5 genes that compose a heterodimer of polyamine transport were modulated. Slc3a2 expression increased in macrophages supplemented with putrescine or L-arginine and spermidine during infection and Slc7a5 increased in spermidine or spermine supplementation during infection. The putrescine supplementation of macrophages during infection increased monocyte chemoattractant protein (Mcp1) gene expression. The infection analysis showed that supplementation with putrescine, L-arginine plus spermidine, or L-arginine plus spermine reduced the percentage of infected macrophage compared to L-arginine supplementation condition. Our data suggest that the polyamines regulate the expression of genes related to their transport and metabolism macrophages infected with 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

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

Relative expression of proinflammatory cytokines in BALB/c macrophages infected or not with <i>L</i>. <i>amazonensis</i> with or without polyamines supplementation.

Macrophages were supplemented with putrescine (put+), spermidine (spd+), spermine (spm+) with or without L-arginine (arg+) concomitant or not to L. amazonensis infection (MOI 5:1) for 4h, and after 24h in complete medium. RNA was extracted for cDNA conversion and relative quantification of genes Il-1b (A) and Mcp1 (B) by RT-qPCR. Data were normalized using the β-2-microglobulin gene, and uninfected macrophage arg+ at 4 h was used as a reference for ΔΔCT relative quantification. The bars represent the averages and S.E.M. We performed three independent experiments. Statistical analysis using One-Way ANOVA with mixed-effects, post-hoc test Sidak’s multiple comparisons. #: p≤0.05 for the comparison between 4h vs. 24h.</p

Correlation and heat map analysis.

(A) PCA analysis of gene expression variance in all conditions analyzed after 4h in infected and non-infected macrophages and (B) percentage of contributions of each gene to the Principal Component 1 (PC1). (C) The Heat Map analysis of the Log2-fold change of genes upregulated (red) and downregulated (blue) and (D) gene expression correlation matrix (Spearman correlation) showed positive (orange) and negative (blue) associations between gene expression from data of macrophages supplemented with putrescine (put+), spermidine (spd+), spermine (spm+) with or without L-arginine (arg+) concomitant or not to L. amazonensis infection for 4h.</p

List of oligonucleotide pairs.

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.</div

Relative expression of <i>Nos2</i> and NO production in BALB/c macrophages infected or not with <i>L</i>. <i>amazonensis</i> with or without polyamines supplementation.

Macrophages (5x106) (A) and (1x106) (B, C) were supplemented with putrescine (put+), spermidine (spd+), spermine (spm+) with or without L-arginine (arg+) concomitant to L. amazonensis infection (MOI 5:1) for 4h, and after 24h in complete medium. The RNA was extracted for cDNA conversion and relative quantification of genes Nos2 (A) by RT-qPCR. Data were normalized using the β-2-microglobulin gene, and the uninfected macrophage arg+ at 4 h was used as a reference for ΔΔCT relative quantification. The samples were stained with DAF-FM for flow cytometry analysis of DAF-FM+ cells (B). (C) Representative dot plot of DAF-FM detection in macrophages uninfected or infected with L. amazonensis supplemented with arg+ and controls unlabeled or stimulated with LPS plus IFN-γ. (D) Correlation of Nos2 and NO levels. The bars represent the averages and S.E.M. We performed three independent experiments. Statistical analysis using One-Way ANOVA with mixed-effects, post-hoc test Sidak’s multiple comparisons. #: p≤0.05 for the comparison between 4h vs. 24h.</p

Effect of polyamine supplementation in infection of BALB/c macrophages with <i>L</i>. <i>amazonensis</i>.

Macrophages (2x105) were deprived (arg-) or supplemented (arg+) with L-arginine and/or putrescine (put+), spermidine (spd+), spermine (spm+) concomitant to L. amazonensis infection (MOI 5:1) for 4h, and after 24 and 48h in complete medium. Cells were stained using Panoptic to determine the percentage of infected macrophage (A) and the number of amastigotes per macrophage (B). Each box represents the mean ± S.E.M. of 3 independent experiments (n = 500 macrophages). Statistical analysis using Two-Way ANOVA with mixed-effects, post hoc test Sidak’s multiple comparisons: a, p≤0,05 comparing to arg+; b, p≤0,05 comparing to arg+/put+ and put+.</p

Effects of L-arginine and polyamines supplementation in gene expression in BALB/c macrophage during infection with <i>L</i>. <i>amazonensis</i>.

Genes modulated by putrescine, spermidine, spermine, or L-arginine. The L-glutamine transporter SLC1A5 is shown in the mitochondrial membrane. SLC25A15 performs the L-citrulline and L-ornithine antiport between the mitochondria and cytosol. The enzymes from polyamines biosynthesis: arginase 1 (ARG1) converts L-arginine into ornithine, which is subsequently converted by ornithine decarboxylase 1 (ODC1) into putrescine. Putrescine is converted by spermidine synthase (SPDS) into spermidine; spermidine is converted by spermine synthase (SPMS) into spermine. The Nitric oxide 2 (NOS2) enzyme uses L-arginine to produce nitric oxide (NO) and citrulline. Also, mitochondrial arginase 2 (ARG2) uses L-arginine to produce ornithine. E, extracellular environment. The L-arginine transporters CAT1 and CAT2 and the heterodimeric amino acid and polyamines transporter SLC3A2/SLC7A5 are shown in the plasmatic membrane. The amino acid and polyamines transporters can also mediate transport in the phagolysosome membrane. Created with Biorender.com.</p

Relative expression of polyamine biosynthesis-related genes in BALB/c macrophages infected or not with <i>L</i>. <i>amazonensis</i> with polyamine supplementation.

Macrophages were supplemented with putrescine (put+), spermidine (spd+), spermine (spm+) with or without L-arginine (arg+) concomitant to L. amazonensis infection (MOI 5:1) for 4h, and after 24h in complete medium. The RNA was extracted for cDNA conversion and relative quantification of genes Arg1 (A), Arg2 (B) and Odc1 (C) by RT-qPCR. Data were normalized using the β-2-microglobulin gene, and the uninfected macrophage arg+ at 4 h was used as reference in ΔΔCT relative quantification. The bars represent the averages and S.E.M. We performed three independent experiments. Statistical analysis using One-Way ANOVA with mixed-effects, post-hoc test Sidak’s multiple comparisons.</p