11 research outputs found

    The role of 5-lipoxygenase-derived lipid mediators during the experimental Trypanosoma cruzi infection in mice

    No full text
    No presente trabalho verificamos o papel dos Leucotrienos na modulação da resposta imune durante a fase aguda da infecção experimental pelo Trypanosoma cruzi, usando como modelo camundongos deficientes da enzima 5-lipoxigenase (5-LOko). Os nossos dados demonstram que camundongos infectados pelo T. cruzi produzem metabólitos do ácido aracdônico como PGE2, LTB4 e LTC4. Comparados aos animais controles, os animais 5-LOko apresenta parasitemia mais tardia e menor, tem menor parasitismo tissular, menor infiltrado de células inflamatórias no coração e musculatura esquelética e apresenta menor taxa de mortalidade durante a fase aguda, indicando que animais deficientes de leucotrienos são mais resistentes a infecção pelo parasito. Animais 5-LOko está relacionado com a manutenção de números elevados de células F4/80+ e redução de células CD11b+ durante a infecção e menor número de células T ativadas expressando os marcadores CD4+CD69+, CD4+CD25+, CD4+CD44+ e CD8+CD69+, números inalterados de células T regulatórias CD4+CD25+GITR+ e menor produção de anticorpos parasito-específicos do isotipo IgG2a. O controle eficiente de parasitas por animais 5-LOko está associado ao aumento de células Gr-1+ e CD11c+GR-1+, produção aumentada IL-12, IFN-g, e produzirem menos PGE2, IL-10, ao contrario, animais controles, incapazes de controlar parasitas circulantes, produzem mais PGE2 e IL-10 e menos IL-12 e IFN-g. A baixa mortalidade de animais 5-LOko correlaciona com a produção de PGE2 e IL-10, produzir muita IL-12 e menos IFN-g e NO e baixíssima parasitemia. A mortalidade maior de animais controles envolve a produção IFN-g e altos níveis de LTB4, LTC4, NO e ausência de IL-10, IL-1b, PGE2 e números elevados de parasitas circulantes. Ainda macrófagos de animais 5-LOko apresentam maior capacidade de adesão/internalização de tripomastigotas e alta atividade tripanocida por mecanismo independente da geração de NO. Estes dados em conjunto demonstram que mediadores lipídicos produzidos pela enzima 5-lipoxigenase como LTB4 e LTC4 modulam negativamente a capacidade dos camundongos para geração de uma resposta imune capaz de controlar os parasitos durante a fase aguda da infecção pelo T. cruzi.Accumulating studies have indicated that 5-lipoxigenase (5-LO) converted lipid mediators as leukotrienes acts modulating the host immune response against infectious diseases. The precise role of leukotrienes during the protozoan infection is unknown. In this work we evaluate the role of leukotrienes during the acute phase of Trypanosoma cruzi infection using as model the 5-lypoxigenase deficient mice (5-LOko). Our results show that PGE2, LTB4 and LTC4 are produced during the Trypanosoma cruzi infection. 5-LOko infected mice are more resistant than control mice as judge by the lower parasitemia, decreased number of parasite nest and inflammatory cells in the heart and skeletal muscle and low rate of mortality. The resistance of 5-LOko mice is associated with the increased capacity of spleen cells to produce cytokines as IL-12 and IFN-g; sustained capacity to produce detectable levels of IL-10 and PGEe and low NO serum levels than control mice. In contrast, the wild type mice are extremely susceptible and are unable to control parasites efficiently. The susceptibility is associated with increased levels of IL-10 and PGE2 and low IL-12 and IFN-g production. The high mortality rate in wild type mice is related with high LTB4, LTC4 and NO levels and bias to produce only type 1 cytokines. Also we shown that resistant 5-LOko mice present increased number of spleen cells expressing GR-1+, GR-1+CD11c+, F4/80+ and lower numbers o spleen cells expressing CD4+CD69+, CD4+CD25+, CD4+CD44+, CD8+CD69+ and CD11b+ and low serum levels of parasite-specific IgG2a than wild type mice and do not present alteration in TREG expressing CD4+CD25+GITR+. Importantly, IFN-g and- LPS activated macrophage from 5-LOko mice but not from wild type mice, present high capacity to recognize typomastigotes, internalize them and strong capacity to kill intracellular parasite as NO independent pathway. The results implicate that high levels leukotrienes, NO and pure type 1 cytokines production is associated to susceptibility to parasite. In contrast, leukotrienes deficiency led to an balanced immune response with relative high levels of type 1 cytokines and relative low levels of NO, type 2 cytokines and PGE2 that efficiently control the parasites. Also indicate that 5-lipoxigenase converted lipid mediators contribute negatively to generation of an effective immune response during the acute phase of T. cruzi infection

    The role of 5-lipoxygenase-derived lipid mediators during the experimental Trypanosoma cruzi infection in mice

    No full text
    No presente trabalho verificamos o papel dos Leucotrienos na modulação da resposta imune durante a fase aguda da infecção experimental pelo Trypanosoma cruzi, usando como modelo camundongos deficientes da enzima 5-lipoxigenase (5-LOko). Os nossos dados demonstram que camundongos infectados pelo T. cruzi produzem metabólitos do ácido aracdônico como PGE2, LTB4 e LTC4. Comparados aos animais controles, os animais 5-LOko apresenta parasitemia mais tardia e menor, tem menor parasitismo tissular, menor infiltrado de células inflamatórias no coração e musculatura esquelética e apresenta menor taxa de mortalidade durante a fase aguda, indicando que animais deficientes de leucotrienos são mais resistentes a infecção pelo parasito. Animais 5-LOko está relacionado com a manutenção de números elevados de células F4/80+ e redução de células CD11b+ durante a infecção e menor número de células T ativadas expressando os marcadores CD4+CD69+, CD4+CD25+, CD4+CD44+ e CD8+CD69+, números inalterados de células T regulatórias CD4+CD25+GITR+ e menor produção de anticorpos parasito-específicos do isotipo IgG2a. O controle eficiente de parasitas por animais 5-LOko está associado ao aumento de células Gr-1+ e CD11c+GR-1+, produção aumentada IL-12, IFN-g, e produzirem menos PGE2, IL-10, ao contrario, animais controles, incapazes de controlar parasitas circulantes, produzem mais PGE2 e IL-10 e menos IL-12 e IFN-g. A baixa mortalidade de animais 5-LOko correlaciona com a produção de PGE2 e IL-10, produzir muita IL-12 e menos IFN-g e NO e baixíssima parasitemia. A mortalidade maior de animais controles envolve a produção IFN-g e altos níveis de LTB4, LTC4, NO e ausência de IL-10, IL-1b, PGE2 e números elevados de parasitas circulantes. Ainda macrófagos de animais 5-LOko apresentam maior capacidade de adesão/internalização de tripomastigotas e alta atividade tripanocida por mecanismo independente da geração de NO. Estes dados em conjunto demonstram que mediadores lipídicos produzidos pela enzima 5-lipoxigenase como LTB4 e LTC4 modulam negativamente a capacidade dos camundongos para geração de uma resposta imune capaz de controlar os parasitos durante a fase aguda da infecção pelo T. cruzi.Accumulating studies have indicated that 5-lipoxigenase (5-LO) converted lipid mediators as leukotrienes acts modulating the host immune response against infectious diseases. The precise role of leukotrienes during the protozoan infection is unknown. In this work we evaluate the role of leukotrienes during the acute phase of Trypanosoma cruzi infection using as model the 5-lypoxigenase deficient mice (5-LOko). Our results show that PGE2, LTB4 and LTC4 are produced during the Trypanosoma cruzi infection. 5-LOko infected mice are more resistant than control mice as judge by the lower parasitemia, decreased number of parasite nest and inflammatory cells in the heart and skeletal muscle and low rate of mortality. The resistance of 5-LOko mice is associated with the increased capacity of spleen cells to produce cytokines as IL-12 and IFN-g; sustained capacity to produce detectable levels of IL-10 and PGEe and low NO serum levels than control mice. In contrast, the wild type mice are extremely susceptible and are unable to control parasites efficiently. The susceptibility is associated with increased levels of IL-10 and PGE2 and low IL-12 and IFN-g production. The high mortality rate in wild type mice is related with high LTB4, LTC4 and NO levels and bias to produce only type 1 cytokines. Also we shown that resistant 5-LOko mice present increased number of spleen cells expressing GR-1+, GR-1+CD11c+, F4/80+ and lower numbers o spleen cells expressing CD4+CD69+, CD4+CD25+, CD4+CD44+, CD8+CD69+ and CD11b+ and low serum levels of parasite-specific IgG2a than wild type mice and do not present alteration in TREG expressing CD4+CD25+GITR+. Importantly, IFN-g and- LPS activated macrophage from 5-LOko mice but not from wild type mice, present high capacity to recognize typomastigotes, internalize them and strong capacity to kill intracellular parasite as NO independent pathway. The results implicate that high levels leukotrienes, NO and pure type 1 cytokines production is associated to susceptibility to parasite. In contrast, leukotrienes deficiency led to an balanced immune response with relative high levels of type 1 cytokines and relative low levels of NO, type 2 cytokines and PGE2 that efficiently control the parasites. Also indicate that 5-lipoxigenase converted lipid mediators contribute negatively to generation of an effective immune response during the acute phase of T. cruzi infection

    Amastin Knockdown in <i>Leishmania braziliensis</i> Affects Parasite-Macrophage Interaction and Results in Impaired Viability of Intracellular Amastigotes

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    <div><p>Leishmaniasis, a human parasitic disease with manifestations ranging from cutaneous ulcerations to fatal visceral infection, is caused by several <i>Leishmania</i> species. These protozoan parasites replicate as extracellular, flagellated promastigotes in the gut of a sandfly vector and as amastigotes inside the parasitophorous vacuole of vertebrate host macrophages. Amastins are surface glycoproteins encoded by large gene families present in the genomes of several trypanosomatids and highly expressed in the intracellular amastigote stages of <i>Trypanosoma cruzi</i> and <i>Leishmania</i> spp. Here, we showed that the genome of <i>L</i>. <i>braziliensis</i> contains 52 amastin genes belonging to all four previously described amastin subfamilies and that the expression of members of all subfamilies is upregulated in <i>L</i>. <i>braziliensis</i> amastigotes. Although primary sequence alignments showed no homology to any known protein sequence, homology searches based on secondary structure predictions indicate that amastins are related to claudins, a group of proteins that are components of eukaryotic tight junction complexes. By knocking-down the expression of δ-amastins in <i>L</i>. <i>braziliensis</i>, their essential role during infection became evident. δ-amastin knockdown parasites showed impaired growth after <i>in vitro</i> infection of mouse macrophages and completely failed to produce infection when inoculated in BALB/c mice, an attenuated phenotype that was reverted by the re-expression of an RNAi-resistant amastin gene. Further highlighting their essential role in host-parasite interactions, electron microscopy analyses of macrophages infected with amastin knockdown parasites showed significant alterations in the tight contact that is normally observed between the surface of wild type amastigotes and the membrane of the parasitophorous vacuole.</p></div

    Amastin knockdown affects amastigote interactions with macrophage membranes.

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    <p>(A) Transmission electron microscopy of macrophages infected with WT <i>L</i>. <i>braziliensis</i> (WT) and two cloned cell lines expressing amastin siRNA, RNAi-1060 cl1 and RNAi-1060 cl5. Left side panels show amastigotes surrounded by the macrophage parasitophorous vacuole (PV) membrane. In contrast to WT parasites, that are in close contact with the PV membrane, larger distances between the membranes of intracellular amastigotes and the PV membranes are observed in macrophages infected with both clones of RNAi knockdown mutants. Right side panels show magnified images of the points of contact between parasite (arrows) and PV (arrowheads) membranes with regions where disrupted membrane structures are observed in macrophages infected with the RNAi knockdown mutants. Parasite nucleus (n), kinetoplast (k), subpellicular microtubule (m) and flagellum (f) are indicated by lower case letters. (B) Ploted ratios between the area of macrophage PV and the area of the amastigote inside each vacuole. Error bars represented the SD values obtained from measurements of the areas of twenty vacuoles and their respective parasites in each experimental group.</p

    Phylogenetic tree of amino acid sequences of 52 amastins from <i>L</i>. <i>braziliensis</i>.

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    <p>The sequences were aligned using the MUSCLE algorithm and a neighbor joining tree was generated using the MEGA6 software. Branch lengths are drawn proportion to evolutionary change with bootstrap values shown on each node. Classification into four amastin sub-families shown on the right was based on Jackson (2010).</p

    Infection of mouse macrophages and BALB/c mice footpads with WT <i>L</i>. <i>braziliensis</i> and RNAi-1060 cell lines.

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    <p>(A) Intraperitoneal macrophages from BALB/c mice were incubated for 24 hours at 34°C with stationary phase promastigotes from WT <i>L</i>. <i>braziliensis</i> cultures and the two cloned cell lines RNAi-Ama1060 cl1 and cl5 at a ratio of 10: 1 parasites/cell. After washing non-internalized promastigotes macrophages were incubated for 24, 48 or 72 hours before the cells were stained with DAPI and the numbers of intracellular amastigotes, visualized by fluorescence microscopy, were determined. (B) BALB/c mice were infected in the footpads with 10<sup>7</sup> stationary phase promastigotes from WT <i>L</i>. <i>braziliensis</i>, <i>L</i>. <i>braziliensis</i> transfected with the pIR1PHLEO vector containing GFP, and two cloned cell lines expressing δ-amastin siRNA, RNAi-1060cl1 and cl5. Two, four and nine weeks after infection, parasitism was evaluated by the limiting dilution method.</p

    Differential expression of amastin mRNA during the <i>L</i>. <i>braziliensis</i> life cycle.

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    <p>Total RNA (10 μg/lane), extrated from promatigote (P) and axenic amastigote (A) forms were separated by electrophoresis, transfered to nylon membranes and probed with the <sup>32</sup>P-labelled sequences corresponding to an α-amastin (LbM.28.1550), β-amastin (LbM.30.0980), γ-amastin (LbrM.24.1600) and two δ-amastins (LbM.08.0300 and LbrM.20.1060). Bottom panels show hybridization of the same membranes with a fragment of the 5S rRNA.</p

    RNAi knockdown of amastin genes in <i>Leishmania braziliensis</i>.

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    <p>(A) The plR1-Phleo plasmid containing two opposite amastin fragments with a stem-loop stuffer fragment (black box), Phleomycin resistance (gene PHLEO) and the rRNA promoter (P rRNA) is shown integrated into the SSU rRNA locus of <i>Leishmania</i> (gray box). (B) Northern blot analyses of RNA isolated from (P) promastigote and (A) axenic amastigotes from wild type <i>L</i>. <i>braziliensis</i> (WT) and two cloned cell lines of <i>L</i>. <i>braziliensis</i> transfected with a construct that generates δ-amastin dsRNA named RNAi-1060-cl1 and cl5. The blots were probed with a <sup>32</sup>P-labelled DNA fragment corresponding to the LbrM.08.1060 amastin gene. (C) Low-molecular-weight RNAs isolated from promastigotes and amastigotes from WT <i>L</i>. <i>braziliensis</i> as well as from promastigotes from RNAi-1060 cl1 and amastigotes from the two cloned cell line transfected with amastin dsRNA constructs (RNAi-1060 cl1 and cl5) were fractionated on a 15% polyacrylamide gel and probed with a mixture of <sup>32</sup>P-labelled oligonucleotide probes corresponding to the full length LbrM.08.1060 amastin gene. siRNA indicates the position of small interfering RNA bands that hybridized with δ-amastin oligonucleotide probes, which co-migrate with a 26 nt DNA molecular weight marker. Hybridization of the same blot with a probe corresponding to the <i>L</i>. <i>braziliensis</i> Glu-tRNA is also shown as a loading control. (D) Total protein extracts from the cloned cell RNAi-1060 cl1 was analyzed by western blot using an antibody generated against the recombinant Ama1060. The same blot was incubated with anti-α-tubulin as a loading control.</p

    Homology-based 3D modeling of α, β, γ and δ amastins.

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    <p>Structural predictions were done using PHYRE web server and the predicted structures of α, β, γ and δ amastins, were imaged using the UCFS Chimera program. α-amastin is shown in green, β-amastin is shown in gray, γ-amastin is shown in red, δ-amastin is shown in yellow and the superimposed mouse claudin 15 model is shown in blue.</p

    Subcellular localization of distinct amastins in fusion with GFP.

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    <p>Promastigotes were transiently transfected with the plasmids pSP-Ama0980-GFP (A), pSP-Ama1600-GFP (B), pSP-Ama1060-GFP (C) and pSP-nGFP (D) as a control plasmid. Transfected parasites were fixed with 2% paraformaldehyde, stained with DAPI and visualized under a fluorescence microscope. Nuclear and kinetoplast DNA are shown in blue.</p
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