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Pyrimidinergic Receptor Activation Controls Toxoplasma gondii Infection in Macrophages.

Moreira-Souza AC, Marinho Y, Correa G, Santoro GF, Coutinho CM, Vommaro RC, Coutinho-Silva R - PLoS ONE (2015)

Bottom Line: Treatment with these nucleotides reduced parasitic load by 90%, but did not increase the levels of the inflammatory mediators NO and ROS, nor did it modulate host cell death by apoptosis or necrosis.In subsequent infections, prematurely egressed parasites had reduced infectivity, and could neither replicate nor inhibit the fusion of lysosomes to the parasitophorous vacuole.The use of selective agonists and antagonists of the receptor subtypes P2Y2 and P2Y4 and P2Y6 showed that premature parasite egress may be mediated by the activation of these receptor subtypes.

View Article: PubMed Central - PubMed

Affiliation: Laboratório de Imunofisiologia, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, 21941-902, Brazil; Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, 21941-902, Brazil; Instituto Nacional de Ciência e Tecnologia para Pesquisa Translacional em Saúde e Ambiente na Região Amazônica (INPeTAm/UFRJ), Rio de Janeiro, RJ, 21941-902, Brazil.

ABSTRACT
Infection by the protozoan parasite Toxoplasma gondii is highly prevalent worldwide and may have serious clinical manifestations in immunocompromised patients. T. gondii is an obligate intracellular parasite that infects almost any cell type in mammalian hosts, including immune cells. The immune cells express purinergic P2 receptors in their membrane--subdivided into P2Y and P2X subfamilies--whose activation is important for infection control. Here, we examined the effect of treatment with UTP and UDP in mouse peritoneal macrophages infected with T. gondii tachyzoites. Treatment with these nucleotides reduced parasitic load by 90%, but did not increase the levels of the inflammatory mediators NO and ROS, nor did it modulate host cell death by apoptosis or necrosis. On the other hand, UTP and UDP treatments induced early egress of tachyzoites from infected macrophages, in a Ca2+-dependent manner, as shown by scanning electron microscopy analysis, and videomicroscopy. In subsequent infections, prematurely egressed parasites had reduced infectivity, and could neither replicate nor inhibit the fusion of lysosomes to the parasitophorous vacuole. The use of selective agonists and antagonists of the receptor subtypes P2Y2 and P2Y4 and P2Y6 showed that premature parasite egress may be mediated by the activation of these receptor subtypes. Our results suggest that the activity of P2Y host cell receptors controls T. gondii infection in macrophages, highlighting the importance of pyrimidinergic signaling for innate immune system response against infection. Finally the P2Y receptors should be considered as new target for the development of drugs against T. gondii infection.

No MeSH data available.


Related in: MedlinePlus

P2Y receptor agonists induce calcium-dependent premature egress of tachyzoites from host cells.Macrophages infected with T. gondii tachyzoites at a ratio of 5:1 parasites per host cell were treated for 30 minutes with 100 μM UTP or UDP, or for 15 minutes with 5 μM of the Ca2+ ionophore 4Bra23187. Also, some samples were pre-incubated with 2 mM of the calcium chelator BAPTA-AM or with 2 μM of the phospholipase C inhibitor U73122 for 30 minutes, before UTP treatment. Cells were processed for light microscopy analysis of the infection index (A and C), and the number of free tachyzoites in culture supernatants was determined using a hemocytometer (B and D). Infected macrophages (M) were treated with 100 μM of UTP or UDP for 15 minutes and then processed for scanning electronic microscopy. The images show parasites activelly egressing from host cells (E and F), with extruded conoids (arrows). (A, B, C) Data represent mean and SEM of ten (A), seven (B) and three (C) independent experiments. (D) Data are representative of two independent experiments. ** p<0.001 and *** p < 0.0001 relative to untreated. ## p<0.01 and ### p<0.0001 relative to treated in presence of phospholipase C inhibitor.
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pone.0133502.g003: P2Y receptor agonists induce calcium-dependent premature egress of tachyzoites from host cells.Macrophages infected with T. gondii tachyzoites at a ratio of 5:1 parasites per host cell were treated for 30 minutes with 100 μM UTP or UDP, or for 15 minutes with 5 μM of the Ca2+ ionophore 4Bra23187. Also, some samples were pre-incubated with 2 mM of the calcium chelator BAPTA-AM or with 2 μM of the phospholipase C inhibitor U73122 for 30 minutes, before UTP treatment. Cells were processed for light microscopy analysis of the infection index (A and C), and the number of free tachyzoites in culture supernatants was determined using a hemocytometer (B and D). Infected macrophages (M) were treated with 100 μM of UTP or UDP for 15 minutes and then processed for scanning electronic microscopy. The images show parasites activelly egressing from host cells (E and F), with extruded conoids (arrows). (A, B, C) Data represent mean and SEM of ten (A), seven (B) and three (C) independent experiments. (D) Data are representative of two independent experiments. ** p<0.001 and *** p < 0.0001 relative to untreated. ## p<0.01 and ### p<0.0001 relative to treated in presence of phospholipase C inhibitor.

Mentions: Since the reduction in T. gondii macrophage infection induced by P2Y agonist treatment was not due to increased NO or ROS production, or cell death induction, we hypothesized that tachyzoites might be egressing prematurely from infected cells upon nucleotide treatment. To test this hypothesis, we determined the infection index in cultures (2 h post-infection) fixed immediately after treatment with 100 μM UTP or UDP for 30 minutes (Fig 3A), and also quantified the number of parasites observed in the culture medium after treatment (Fig 3B). We observed that both UTP and UDP treatment reduced the number of parasite inside of cells with an overall reduction in parasite burden of 38% and 33%, respectively (Fig 3A). Ca2+ influx into host cells induces T. gondii egress even after a short period of infection [11]. Thus, treatment with the Ca2+ ionophore 4BrA23187 (for 15 min) was used as a positive control for early tachyzoite egress from host cells. Similarly to that observed after treatment with both UTP and UDP treatments 4BrA23187, reduced the number of infected macrophages and the infection index, (Fig 3A).


Pyrimidinergic Receptor Activation Controls Toxoplasma gondii Infection in Macrophages.

Moreira-Souza AC, Marinho Y, Correa G, Santoro GF, Coutinho CM, Vommaro RC, Coutinho-Silva R - PLoS ONE (2015)

P2Y receptor agonists induce calcium-dependent premature egress of tachyzoites from host cells.Macrophages infected with T. gondii tachyzoites at a ratio of 5:1 parasites per host cell were treated for 30 minutes with 100 μM UTP or UDP, or for 15 minutes with 5 μM of the Ca2+ ionophore 4Bra23187. Also, some samples were pre-incubated with 2 mM of the calcium chelator BAPTA-AM or with 2 μM of the phospholipase C inhibitor U73122 for 30 minutes, before UTP treatment. Cells were processed for light microscopy analysis of the infection index (A and C), and the number of free tachyzoites in culture supernatants was determined using a hemocytometer (B and D). Infected macrophages (M) were treated with 100 μM of UTP or UDP for 15 minutes and then processed for scanning electronic microscopy. The images show parasites activelly egressing from host cells (E and F), with extruded conoids (arrows). (A, B, C) Data represent mean and SEM of ten (A), seven (B) and three (C) independent experiments. (D) Data are representative of two independent experiments. ** p<0.001 and *** p < 0.0001 relative to untreated. ## p<0.01 and ### p<0.0001 relative to treated in presence of phospholipase C inhibitor.
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pone.0133502.g003: P2Y receptor agonists induce calcium-dependent premature egress of tachyzoites from host cells.Macrophages infected with T. gondii tachyzoites at a ratio of 5:1 parasites per host cell were treated for 30 minutes with 100 μM UTP or UDP, or for 15 minutes with 5 μM of the Ca2+ ionophore 4Bra23187. Also, some samples were pre-incubated with 2 mM of the calcium chelator BAPTA-AM or with 2 μM of the phospholipase C inhibitor U73122 for 30 minutes, before UTP treatment. Cells were processed for light microscopy analysis of the infection index (A and C), and the number of free tachyzoites in culture supernatants was determined using a hemocytometer (B and D). Infected macrophages (M) were treated with 100 μM of UTP or UDP for 15 minutes and then processed for scanning electronic microscopy. The images show parasites activelly egressing from host cells (E and F), with extruded conoids (arrows). (A, B, C) Data represent mean and SEM of ten (A), seven (B) and three (C) independent experiments. (D) Data are representative of two independent experiments. ** p<0.001 and *** p < 0.0001 relative to untreated. ## p<0.01 and ### p<0.0001 relative to treated in presence of phospholipase C inhibitor.
Mentions: Since the reduction in T. gondii macrophage infection induced by P2Y agonist treatment was not due to increased NO or ROS production, or cell death induction, we hypothesized that tachyzoites might be egressing prematurely from infected cells upon nucleotide treatment. To test this hypothesis, we determined the infection index in cultures (2 h post-infection) fixed immediately after treatment with 100 μM UTP or UDP for 30 minutes (Fig 3A), and also quantified the number of parasites observed in the culture medium after treatment (Fig 3B). We observed that both UTP and UDP treatment reduced the number of parasite inside of cells with an overall reduction in parasite burden of 38% and 33%, respectively (Fig 3A). Ca2+ influx into host cells induces T. gondii egress even after a short period of infection [11]. Thus, treatment with the Ca2+ ionophore 4BrA23187 (for 15 min) was used as a positive control for early tachyzoite egress from host cells. Similarly to that observed after treatment with both UTP and UDP treatments 4BrA23187, reduced the number of infected macrophages and the infection index, (Fig 3A).

Bottom Line: Treatment with these nucleotides reduced parasitic load by 90%, but did not increase the levels of the inflammatory mediators NO and ROS, nor did it modulate host cell death by apoptosis or necrosis.In subsequent infections, prematurely egressed parasites had reduced infectivity, and could neither replicate nor inhibit the fusion of lysosomes to the parasitophorous vacuole.The use of selective agonists and antagonists of the receptor subtypes P2Y2 and P2Y4 and P2Y6 showed that premature parasite egress may be mediated by the activation of these receptor subtypes.

View Article: PubMed Central - PubMed

Affiliation: Laboratório de Imunofisiologia, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, 21941-902, Brazil; Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, 21941-902, Brazil; Instituto Nacional de Ciência e Tecnologia para Pesquisa Translacional em Saúde e Ambiente na Região Amazônica (INPeTAm/UFRJ), Rio de Janeiro, RJ, 21941-902, Brazil.

ABSTRACT
Infection by the protozoan parasite Toxoplasma gondii is highly prevalent worldwide and may have serious clinical manifestations in immunocompromised patients. T. gondii is an obligate intracellular parasite that infects almost any cell type in mammalian hosts, including immune cells. The immune cells express purinergic P2 receptors in their membrane--subdivided into P2Y and P2X subfamilies--whose activation is important for infection control. Here, we examined the effect of treatment with UTP and UDP in mouse peritoneal macrophages infected with T. gondii tachyzoites. Treatment with these nucleotides reduced parasitic load by 90%, but did not increase the levels of the inflammatory mediators NO and ROS, nor did it modulate host cell death by apoptosis or necrosis. On the other hand, UTP and UDP treatments induced early egress of tachyzoites from infected macrophages, in a Ca2+-dependent manner, as shown by scanning electron microscopy analysis, and videomicroscopy. In subsequent infections, prematurely egressed parasites had reduced infectivity, and could neither replicate nor inhibit the fusion of lysosomes to the parasitophorous vacuole. The use of selective agonists and antagonists of the receptor subtypes P2Y2 and P2Y4 and P2Y6 showed that premature parasite egress may be mediated by the activation of these receptor subtypes. Our results suggest that the activity of P2Y host cell receptors controls T. gondii infection in macrophages, highlighting the importance of pyrimidinergic signaling for innate immune system response against infection. Finally the P2Y receptors should be considered as new target for the development of drugs against T. gondii infection.

No MeSH data available.


Related in: MedlinePlus