Educomunicação e suas áreas de intervenção: Novos paradigmas para o diálogo intercultural

oai:omp.abpeducom.org.br:publicationFormat/1O material aqui divulgado representa, em essência, a contribuição do VII Encontro Brasileiro de Educomunicação ao V Global MIL Week, da UNESCO, ocorrido na ECA/USP, entre 3 e 5 de novembro de 2016. Estamos diante de um conjunto de 104 papers executivos, com uma média de entre 7 e 10 páginas, cada um. Com este rico e abundante material, chegamos ao sétimo e-book publicado pela ABPEducom, em seus seis primeiros anos de existência. A especificidade desta obra é a de trazer as “Áreas de Intervenção” do campo da Educomunicação, colocando-as a serviço de uma meta essencial ao agir educomunicativo: o diálogo intercultural, trabalhado na linha do tema geral do evento internacional: Media and Information Literacy: New Paradigms for Intercultural Dialogue

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

Detection of multiple circulating Leishmania species in Lutzomyia longipalpis in the city of Governador Valadares, southeastern Brazil.

Leishmaniasis encompasses a group of diverse clinical diseases caused by protozoan parasites of the Leishmania genus. This disease is a major public health problem in the New World affecting people exposed in endemic regions. The city of Governador Valadares (Minas Gerais/Brazil) is a re-emerging area for visceral leishmaniasis, with 191 human cases reported from 2008 to 2017 and a lethality rate of 14.7%. The transmission of the parasite occurs intensely in this region with up to 22% of domestic dogs with positive serology for the visceral form. Lu. longipalpis is one of the most abundant sand fly species in this area. Despite this scenario, so far there is no information regarding the circulating Leishmania species in the insect vector Lutzomyia longipalpis in this focus. We collected 616 female Lutzomyia longipalpis sand flies between January and September 2015 in the Vila Parque Ibituruna neighborhood (Governador Valadares/MG), which is located on a transitional area between the sylvatic and urban environments with residences built near a preserved area. After DNA extraction of individual sand flies, the natural Leishmania infections in Lu. longipalpis were detected by conventional PCR, using primers derived from kDNA sequences, specific for L. (Leishmania) or L. (Viannia) subgenus. The sensitivity of these PCR reactions was 0.1 pg of DNA for each Leishmania subgenus and the total infection rate of 16.2% (100 positive specimens). Species-specific PCR detected the presence of multiple Leishmania species in infected Lu. longipalpis specimens in Governador Valadares, including L. amazonensis (n = 3), L. infantum (n = 28), L. (Viannia) spp. (n = 20), coinfections with L. infantum and L. (Viannia) spp. (n = 5), and L. (Leishmania) spp (n = 44). Our results demonstrate that multiple Leishmania species circulate in Lu. longipalpis in Governador Valadares and reveal a potential increasing risk of transmission of the different circulating parasite species. This information reinforces the need for epidemiological and entomological surveillance in this endemic focus, and the development of effective control strategies against leishmaniasis

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