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Disruption of Purinergic Receptor P2X7 Signaling Increases Susceptibility to Cerebral Toxoplasmosis

  • Aline Cristina Abreu Moreira-Souza
    Affiliations
    Laboratory of Immunophysiology, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil

    Laboratory of Cellular Ultrastructure Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
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  • Thuany Prado Rangel
    Affiliations
    Laboratory of Immunophysiology, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
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  • Sthefani Rodrigues Batista da Silva
    Affiliations
    Laboratory of Immunophysiology, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
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  • Vanessa Ribeiro Figliuolo
    Affiliations
    Laboratory of Immunophysiology, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
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  • Luiz Eduardo Baggio Savio
    Affiliations
    Laboratory of Immunophysiology, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
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  • Felipe Schmitz
    Affiliations
    Laboratory of Neuroprotection and Metabolic Disease, Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Rio Grande do Sul, Brazil
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  • Christina Maeda Takiya
    Affiliations
    Laboratory of Immunopathology, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
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  • Angela T.S. Wyse
    Affiliations
    Laboratory of Neuroprotection and Metabolic Disease, Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Rio Grande do Sul, Brazil
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  • Rossiane Claudia Vommaro
    Correspondence
    Address correspondence to Rossiane Claudia Vommaro, Ph.D., or Robson Coutinho-Silva, Ph.D., Instituto de Biofísica Carlos Chagas Filho, Federal University of Rio de Janeiro, Edifício do Centro de Ciências da Saúde, Bloco G. Av. Carlos Chagas Filho, 373. Cidade Universitária, Ilha do Fundão, Rio de Janeiro, RJ 21941-902, Brazil.
    Affiliations
    Laboratory of Cellular Ultrastructure Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil

    National Institute of Science and Technology in Structural Biology and Bioimaging (CENABIO), Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
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  • Robson Coutinho-Silva
    Correspondence
    Address correspondence to Rossiane Claudia Vommaro, Ph.D., or Robson Coutinho-Silva, Ph.D., Instituto de Biofísica Carlos Chagas Filho, Federal University of Rio de Janeiro, Edifício do Centro de Ciências da Saúde, Bloco G. Av. Carlos Chagas Filho, 373. Cidade Universitária, Ilha do Fundão, Rio de Janeiro, RJ 21941-902, Brazil.
    Affiliations
    Laboratory of Immunophysiology, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
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Open ArchivePublished:January 14, 2019DOI:https://doi.org/10.1016/j.ajpath.2019.01.001
      Toxoplasmosis is a neglected disease that affects millions of individuals worldwide. Toxoplasma gondii infection is an asymptomatic disease, with lethal cases occurring mostly in HIV patients and organ transplant recipients. Nevertheless, atypical strains of T. gondii in endemic locations cause severe pathology in healthy individuals. Toxoplasmosis has no cure but it can be controlled by the proinflammatory immune response. The purinergic receptor P2X7 (P2X7) is involved in many inflammatory events and has been associated with genes that confer resistance against toxoplasmosis in humans. In vitro studies have reported parasite death after P2X7-receptor activation in various cell types. To understand the contribution of P2X7 during cerebral toxoplasmosis, wild-type and P2rx7 knockout mice were infected orally with T. gondii and their pathologic profiles were analyzed. We found that all P2rx7−/− mice died 8 weeks after infection with an increased number of cysts and fewer inflammatory infiltrates in their brains. The cytokines interleukin-1β, interleukin-12, tumor necrosis factor-α, and reactive oxygen species were absent or reduced in P2rx7−/− mice. Taken together, these data suggest that the P2X7 receptor promotes inflammatory infiltrates, proinflammatory cytokines, and reactive oxygen species production in the brain, and that P2X7 signaling mediates major events that confer resistance to cerebral toxoplasmosis.
      Toxoplasmosis is a life-threatening disease caused by Toxoplasma gondii, a protozoan parasite that has access to the central nervous system.
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      Materials and Methods

      Animals, Infections, and Survival Curves

      Female 6- to 8-week-old C57-black/6 (WT) or P2rx7 knockout (P2rx7−/−) mice from Jackson Laboratory (Bar Harbor, ME) were used in this study. All experiments were approved by the Commission for the Ethical Use of Research Animals from the Federal University of Rio de Janeiro (approved protocol number 086/18). Parasites were maintained in carworth farm 1 mice (CF1) mice by oral infection using a maximum volume of 100 μL phosphate-buffered saline (PBS) containing 50 brain cysts of Me-49, a nonvirulent type II T. gondii strain. For experiments, the WT or P2rx7−/− mice were infected orally with five cysts by gavage. Orally infected animals were monitored once a week for 2 months after infection and susceptibility to infection was determined by the death of the animals.

      Quantification of Brain Cysts

      After 30 days of infection, the animals were euthanized and the brains were removed, washed in PBS, and dissociated using 3 mL of cold PBS passing through 18G, 21G, 23G, and 26G syringe needles to obtain brain lysates. Finally, 10 μL of brain lysates were observed under light microscopy to quantify cyst numbers.

      Histology

      Infected or noninfected mice were euthanized 30 days after infection and brains were removed and washed with cold PBS. The brains were fixed in a 10% formalin-zinc buffered solution overnight, dehydrated in ethanol and xylol for three cycles of 30 minutes, and then embedded in paraffin. Paraffin sections (5 μm) were deparaffinized and stained with hematoxylin and eosin.

      Splenocyte Culture and Cytokine Assay

      Spleens were dissociated using a 70-nm cell strainer (BD Biosciences Discovery Labware, Franklin Lakes, NJ) in 1.5 mL ammonium chloride lysis buffer solution, and were maintained on ice for 7 minutes. The cells were washed with 40 mL cold PBS and were centrifuged twice at 2000 × g for 7 minutes. The cells were resuspended in RPMI 1640 medium supplemented with 10% fetal bovine serum (Invitrogen-Gibco, Paisley, Scotland), 2 mmol/L l-glutamine, 50 mmol/L 2-mercaptoethanol, 10 mmol/L HEPES, 1000/L penicillin, and 100 mg/L streptomycin (all from Sigma Chemical Co., St. Louis, MO), and cultivated for 72 hours without stimuli or in the presence of 7 μg/mL of anti-CD3 and 200 μg/mL of anti-CD28 antibody or 10 μg/mL soluble toxoplasma antigen at 37°C in a 5% CO2 humidified incubator. Samples were centrifuged, and the supernatants were used for measuring the concentrations of cytokines TNF-α, IL-1β, IL-2, IL-6, IL-4, IL-10, IFN-γ, and IL-17 using BCA kits (BD). The cytokines IL-1β and IL-12 were measured in brain lysates, obtained as described in Quantification of Brain Cysts, by enzyme-linked immunosorbent assay according to the manufacturer's instructions (R&D Systems, Minneapolis, MN and Peprotech, Rocky Hill, NJ).

      Oxidation Assay DCF

      Brain samples from WT and P2rx7−/− mice, 30 days after infection, were lysed and 60 μL tissue supernatants were incubated at 37°C and protected from light with 100 μmol/L final concentration of H2DCF-DA probe (Invitrogen, CA) in 96-well plates for 30 minutes. The oxidation of the 2′-7′-dichlorofluorescein (DCF) component was measured by fluorometer using wavelengths of 488 nm (excitation) and 520 nm (emission). DCF standards (0.25 to 10 mmol/L) were used to generate a standard curve. The results were expressed as nanomoles of DCF per milligrams of tissue protein.
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      Evaluation of the probe 2',7'-dichlorofluorescin as an indicator of reactive oxygen species formation and oxidative stress.

      SOD and Catalase Assays

      Superoxide dismutase (SOD) activity was measured in the supernatants of brain lysates indirectly by pyrogallol autoxidation inhibition at 420 nm. A standard curve was generated using purified SOD to calculate the SOD activity in the samples. SOD activity was determined by the amount of SOD that inhibited oxidation of epinephrine by 50%, equivalent to 1 unit. The results were expressed as units per milligram of tissue protein.
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      Catalase enzyme activity was assayed in tissue by incubation of brain samples in the reaction media [20 mmol/L H2O2, 0.1% Triton X-100 (Sigma Chemical Co.) in 10 mmol/L potassium phosphate buffer, pH 7.0] and the absorbance decreases were measured at 240 nm.
      • Aebi H.
      Catalase in vitro.
      One micromole of hydrogen peroxide consumed per minute was equivalent to 1 catalase unit. The result was expressed as unit per milligram of tissue protein.

      Hepatic Enzyme Assay

      Cardiac puncture was performed after 30 days of infection, and blood was collected using 0.1% EDTA-treated syringes. Blood samples were centrifuged at 700 × g for 10 minutes. Serum levels of free alanine aminotransferase and aspartate aminotransferase were evaluated using the Transaminase Alanine Aminotransferase Kinetics kit and the Transaminase Aspartate Aminotransferase Kinetics Kit, according to the manufacturer's instructions (Bioclin, Rio de Janeiro, Brazil).

      Statistical Analysis

      A two-tailed t-test was used for comparisons of two groups, whereas multiple comparisons were performed by one-way analysis of variance followed by the Tukey post-test. All statistical analyses were performed using GraphPad Prism 5 (GraphPad, La Jolla, CA).

      Results

      P2X7 Receptor Participates in Cerebral Toxoplasmosis Resistance

      Initially, female animals were orally infected with 10 cysts of the Me-49 strain and were monitored for 30 days to measure survival. During the first 2 weeks after infection, P2rx7−/− mice showed creeping hair, motor impairment, and ocular disease, and then began dying (data not shown). To establish the chronic model of infection, the parasite load was decreased to five cysts per animal. The survival curve after infection with only five cysts showed that the P2rx7−/− group had milder symptoms but died 6 weeks after infection, still showing more susceptibility to toxoplasmosis than the WT group, which showed no signs of infection (Figure 1A). To understand the effect of P2X7 receptor during toxoplasmosis, the disease was analyzed through hepatic enzyme evaluation considering that the liver is a shock organ in acute toxoplasmosis. After infection, both groups had increased levels of transaminases. Alanine aminotransferase and aspartate aminotransferase levels were higher in the P2rx7−/− group than in the WT group (Figure 1, B and C). These results are consistent with the aggressiveness of the infection found in P2rx7−/− animals. To ensure that T. gondii infection progressed to the chronic stage of disease, developing long latent life in the central nervous system, brain lysates were analyzed. Cysts found in brain lysates had different sizes that correlated with the group in which they were isolated (Figure 1D). Interestingly, P2rx7−/− animals presented with significantly smaller cysts than those found in WT mice brain lysates (Figure 1F). Nevertheless, in terms of the number of brain cysts in each group, a greater number of cysts was found in the brain lysates of P2rx7−/− animals than in WT animals (Figure 1E).
      Figure thumbnail gr1
      Figure 1Protection against cerebral toxoplasmosis mediated by the P2X7 receptor. A: Survival curve of 6-week-old female mice orally infected with five cysts of the Me-49 nonvirulent type II Toxoplasma gondii strain. Representative graph of five independent experiments with 7 to 10 animals per group. B and C: A liver damage biomarker, alanine aminotransferase (B), and a biomarker of systemic damage, aspartate aminotransferase (C), were measured in the serum samples of uninfected or T. gondii–infected animals from the wild-type (WT) and P2rx7 knockout (P2rx7−/−) groups. D: Micrographs of cysts found in brain lysates of mice infected for 30 days. E: The number of cysts was quantified 30 days after infection in brain lysates. F: Comparison of P2rx7−/− and WT cyst sizes. Representative data of five independent experiments, with at least five animals per group. ∗∗P < 0.01, ∗∗∗P < 0.001. Scale bars = 50 μm.

      P2X7 Receptor Is Important for the Recruitment of Inflammatory Cells

      During brain analyses, increased organ weight was observed after infection only in WT animals (Figure 2A). This may be due to tissue inflammation because the P2X7 receptor is known to be involved in cell migration during injury and inflammation.
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      The brains were processed for histology to understand the increase in organ weight. The cerebral cortex of uninfected WT mice showed the same morphology as uninfected P2rx7−/− mice (Figure 2B). The infected WT and P2rx7−/− mice (Figure 2B) showed mononuclear inflammatory cells in the meninges (meningitis) that were more prominent in the WT mice (Figure 2B). In the WT mice, it was possible to observe the presence of Toxoplasma cysts, microglial nodules (Figure 2C), and perivascular inflammatory infiltrates (Figure 2C). P2rx7−/−mice showed cysts or ruptured cysts with free bradyzoites in the cerebral parenchyma, with rare recruitment of inflammatory cells (Figure 2C).
      Figure thumbnail gr2
      Figure 2Micrographs of the superficial aspect of the cerebral cortex of wild-type (WT) and P2rx7 knockout (P2rx7−/−) mice. A: Brain weights 30 days after infection. B: Normal meninges in both groups of uninfected mice are shown in the top row. Notice the enlargement of the meninges with intense recruitment of inflammatory cells in the WT mice compared with P2rx7−/− mice (asterisks), and an inflammatory infiltrate that is more pronounced in WT mice brain when compared with P2rx7−/− mice (arrows) in the bottom row. C: In P2rx7−/− mice the cyst numbers are increased compared with WT mice (asterisks). Microglial nodule and prominent mononuclear cell inflammatory foci in WT mice (arrows) are observed, whereas ruptured cysts are present with minimal inflammation or microglial nodules in P2rx7−/− mice. Micrographs of hematoxylin and eosin staining brain sections are representative of five independent experiments with three animals per analysis. ∗∗∗P < 0.001. Scale bars: 20 μm (B); 50 μm (C).

      The P2X7 Receptor Modulates Proinflammatory Cytokines and Microbicidal Events in the Brain during Cerebral Toxoplasmosis

      In the course of infection, signals of morbidity and mortality were observed in P2rx7−/− animals. Ocular disease and motor impairment were the most common manifestations of toxoplasmosis symptoms (data not shown). The effect of the P2X7 receptor was analyzed in innate immune responses inside the brain during cerebral toxoplasmosis. Increased levels of IL-12, IL-1β, TNF-α, and IFN-γ were found in the brain lysates from T. gondii–infected WT mice (Figure 3, A–D). The production of IL-12 was reduced significantly (Figure 3A), whereas IL-1β and TNF-α were absent in infected P2rx7−/− mice (Figure 3, B and C). Similar increased levels of IFN-γ were found in blood samples from T. gondii–infected P2rx7−/− mice when compared with WT groups (Figure 3D).
      Figure thumbnail gr3
      Figure 3Absence of the P2X7 receptor compromises the proinflammatory response in the brain. A–D: Levels of interleukin (IL)-12, IL-1β, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ cytokines in the brain were measured in the supernatants of brain lysates. E: Reactive oxygen species and reactive nitrogen intermediates were determined by detection of DCF levels in the brain. Data are representative of two independent experiments with five animals per group. P < 0.05, ∗∗P < 0.01∗∗∗P < 0.001.
      Because reactive nitrogen intermediates and ROS are associated with parasite death
      • Yarovinsky F.
      Innate immunity to Toxoplasma gondii infection.
      and can be promoted by P2X7 activation during protozoal infections,
      • Chaves S.P.
      • Torres-Santos E.C.
      • Marques C.
      • Figliuolo V.R.
      • Persechini P.M.
      • Coutinho-Silva R.
      • Rossi-Bergmann B.
      Modulation of P2X(7) purinergic receptor in macrophages by Leishmania amazonensis and its role in parasite elimination.
      • Correa G.
      • Marques da S.C.
      • de Abreu Moreira-Souza A.C.
      • Vommaro R.C.
      • Coutinho-Silva R.
      Activation of the P2X(7) receptor triggers the elimination of Toxoplasma gondii tachyzoites from infected macrophages.
      the enzymes involved in the ROS and NO responses in the brain were tested. SOD and catalase activities and ROS production was measured using the H2DCF probe. No differences in catalase and SOD activities were measured in the brains after T. gondii infection (data not shown). Nevertheless, increased amounts of ROS were found only in the brains of WT mice after T. gondii infection. No changes in ROS production in the brains of the P2rx7−/− group were observed after infection (Figure 3E). These results strongly suggest that the P2X7 receptor contributes to the promotion of proinflammatory cytokines and the potent microbicidal mechanism of ROS during cerebral toxoplasmosis.
      The proinflammatory cytokines involved in protection against T. gondii infection also were checked to analyze the systemic impact of P2X7-receptor signaling during cerebral toxoplasmosis. Blood samples showed increased levels of IL-12, IL-1β, and IFN-γ after T. gondii infection in WT mice (Figure 4, A–C). However, in serum from P2rx7−/− mice, the increased levels of IL-12 were reduced when compared with WT samples (Figure 4A), whereas the production of IL-1β was absent in the P2rx7−/− group (Figure 4B). Similarly increased levels of IFN-γ were found in blood samples from T. gondii–infected P2rx7−/− mice when compared with the WT group (Figure 4C).
      Figure thumbnail gr4
      Figure 4The P2X7 receptor contributes to proinflammatory cytokine production during cerebral toxoplasmosis. A–C: Cytokine levels detected in the serum of uninfected or infected wild-type (WT) and P2rx7 knockout (P2rx7−/−) animals. D–G: Cytokine levels detected in the supernatant of splenocyte culture after 72 hours of CD3/CD28 stimulation. H and I: Cytokine levels detected in the supernatant of splenocyte culture after 72 hours of soluble toxoplasma antigen (STAg) stimulation. Data are representative of three independent experiments with 3, 5, and 10 animals per group. P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.
      In the supernatants of splenocyte cultures, only the WT infected group showed increased levels of IL-2, IL-6, and IL-12 in the presence of nonspecific stimuli (CD3/CD28) (Figure 4, D–F). Similarly increased levels of IFN-γ were observed in the supernatants of splenocyte cultures in both infected groups (Figure 4G). An increase in IFN-γ and IL-12 levels was observed in cell cultures from the infected WT group in the presence of soluble toxoplasma antigen stimuli (Figure 4, H and I). However, the increase in IFN-γ level was reduced in P2rx7−/− mice when compared with the WT group (Figure 4I), whereas the production of IL-12 was absent in the P2rx7−/− group (Figure 4H). These results suggest that P2X7 promotes the production of innate proinflammatory cytokines systemically in response to T. gondii chronic infection.

      Discussion

      In endemic countries, Toxoplasma gondii presents high genotype diversity and is associated with lethal cerebral and ocular disease. It is postulated in the literature that under the influence of the immune system, the parasite responds to infection in a proinflammatory manner, with the conversion of the tachyzoites into bradyzoites and cysts.
      • Skariah S.
      • McIntyre M.K.
      • Mordue D.G.
      Toxoplasma gondii: determinants of tachyzoite to bradyzoite conversion.
      Parasite cysts are immunogenically resistant and are found primarily in the smooth muscle and central nervous system, where they persist during the life of the host.
      The P2X7 receptor is an important component during T. gondii infection. A previous study showed increased susceptibility to toxoplasmosis in human individuals who had low responses to P2X7-receptor activation.
      • Jamieson S.E.
      • Peixoto-Rangel A.L.
      • Hargrave A.C.
      • Roubaix L.A.
      • Mui E.J.
      • Boulter N.R.
      • Miller E.N.
      • Fuller S.J.
      • Wiley J.S.
      • Castellucci L.
      • Boyer K.
      • Peixe R.G.
      • Kirisits M.J.
      • Elias L.S.
      • Coyne J.J.
      • Correa-Oliveira R.
      • Sautter M.
      • Smith N.C.
      • Lees M.P.
      • Swisher C.N.
      • Heydemann P.
      • Noble A.G.
      • Patel D.
      • Bardo D.
      • Burrowes D.
      • McLone D.
      • Roizen N.
      • Withers S.
      • Bahia-Oliveira L.M.
      • McLeod R.
      • Blackwell J.M.
      Evidence for associations between the purinergic receptor P2X(7) (P2RX7) and toxoplasmosis.
      In an acute model of infection, it recently was reported that the absence of functional P2X7 receptor induced severe intestinal disease, ileitis,
      • Miller C.M.
      • Zakrzewski A.M.
      • Robinson D.P.
      • Fuller S.J.
      • Walker R.A.
      • Ikin R.J.
      • Bao S.J.
      • Grigg M.E.
      • Wiley J.S.
      • Smith N.C.
      Lack of a functioning P2X7 receptor leads to increased susceptibility to toxoplasmic ileitis.
      and compromised the production of the key cytokines in the very early phase of infection.
      • Correa G.
      • Almeida L.C.
      • Moreira-Souza A.C.
      • Savio L.E.
      • Takiya C.M.
      • Marques-da-Silva C.
      • Vommaro R.C.
      • Coutinho-Silva R.
      Inflammatory early events associated to the role of P2X7 receptor in acute murine toxoplasmosis.
      In the present study, P2rx7−/− animals showed severe disease after infection with various parasite loads, and some of the group died 2 weeks after infection (ie, during the acute phase). The functions of P2X7 during inflammation involve many cellular events, including cellular activation, cellular migration, cellular death, cytokine and chemokine secretion, and diverse stimuli.
      • Di V.F.
      • Dal B.D.
      • Sarti A.C.
      • Giuliani A.L.
      • Falzoni S.
      The P2X7 receptor in infection and inflammation.
      In an acute model of T. gondii infection, the absence of the P2X7 receptor compromised the proinflammatory response, leading to free dissemination of this parasite and multiple organ failures.
      • Correa G.
      • Almeida L.C.
      • Moreira-Souza A.C.
      • Savio L.E.
      • Takiya C.M.
      • Marques-da-Silva C.
      • Vommaro R.C.
      • Coutinho-Silva R.
      Inflammatory early events associated to the role of P2X7 receptor in acute murine toxoplasmosis.
      In the present study, even in the chronic stage of infection, the P2X7 receptor remained important to promote protection against T. gondii, mediating increased systemic levels of the proinflammatory cytokine IL-1β. The results showed reduced inflammatory infiltrates in the brain and increased markers of acute infection in the absence of the P2X7 receptor. The P2rx7−/− animals presented acute profiles of toxoplasmosis even 30 days after infection, when the infection naturally is focused in the central nervous system.
      The importance of the immune response mediated by cytokines during toxoplasmosis disease has been established.
      • Biswas A.
      • Bruder D.
      • Wolf S.A.
      • Jeron A.
      • Mack M.
      • Heimesaat M.M.
      • Dunay I.R.
      Ly6C(high) monocytes control cerebral toxoplasmosis.
      Proinflammatory cytokines such as IL-12 and IL-1β play crucial roles in control of the parasite,
      • Hitziger N.
      • Dellacasa I.
      • Albiger B.
      • Barragan A.
      Dissemination of Toxoplasma gondii to immunoprivileged organs and role of Toll/interleukin-1 receptor signalling for host resistance assessed by in vivo bioluminescence imaging.
      • Scharton-Kersten T.
      • Denkers E.Y.
      • Gazzinelli R.
      • Sher A.
      Role of IL12 in induction of cell-mediated immunity to Toxoplasma gondii.
      • Sher A.
      • Collazzo C.
      • Scanga C.
      • Jankovic D.
      • Yap G.
      • Aliberti J.
      Induction and regulation of IL-12-dependent host resistance to Toxoplasma gondii.
      and are responsible for potentiating microbicidal mechanisms induced by IFN-γ, including ROS and NO production, lysosomal fusion, and others.
      • Yarovinsky F.
      Innate immunity to Toxoplasma gondii infection.
      The absence of proinflammatory cytokines is associated with the lethality of T. gondii infection.
      • Torres M.
      • Guiton R.
      • Lacroix-Lamande S.
      • Ryffel B.
      • Leman S.
      • Dimier-Poisson I.
      MyD88 is crucial for the development of a protective CNS immune response to Toxoplasma gondii infection.
      During the chronic stage of toxoplasmosis, primarily in the central nervous system, in the absence of immune control, cysts can grow without restriction and bradyzoites eventually exit from the infected cells and initiate new infections, originating new young cysts. These events are associated with toxoplasmosis susceptibility and symptoms are apparent.
      • Silveira C.
      • Vallochi A.L.
      • Rodrigues daSU.
      • Muccioli C.
      • Holland G.N.
      • Nussenblatt R.B.
      • Belfort R.
      • Rizzo L.V.
      Toxoplasma gondii in the peripheral blood of patients with acute and chronic toxoplasmosis.
      • Silveira C.
      • Muccioli C.
      • Nussenblatt R.
      • Belfort Jr., R.
      The effect of long-term intermittent trimethoprim/sulfamethoxazole treatment on recurrences of toxoplasmic retinochoroiditis: 10 years of follow-up.
      P2 signaling in the central nervous system has been reported to contribute to physiologic and pathologic events. The ATP molecule is known as a neurotransmitter that participates in synapses, contributing to physiological brain functions. On the other hand, excessive ATP signaling in the well-controlled microenvironment of the brain can cause damage to the tissue by promoting organ inflammation, cytokine releases, oxidative stress, and neuron cell death, compromising brain function.
      • Savio L.E.
      • Andrade M.G.
      • de Andrade M.P.
      • Santana P.T.
      • Moreira-Souza A.C.
      • Kolling J.
      • Longoni A.
      • Feldbrugge L.
      • Wu Y.
      • Wyse A.T.
      • Robson S.C.
      • Coutinho-Silva R.
      P2X7 receptor signaling contributes to sepsis-associated brain dysfunction.
      • Burnstock G.
      Physiopathological roles of P2X receptors in the central nervous system.
      Nevertheless, the P2 pathway can be disrupted by the parasite during infection. Infection with Me-49 induced a reduction in levels of ATP, ADP, AMP, adenosine, and inosine after 30 days of infection.
      • Tonin A.A.
      • Da Silva A.S.
      • Casali E.A.
      • Silveira S.S.
      • Moritz C.E.
      • Camillo G.
      • Flores M.M.
      • Fighera R.
      • Thome G.R.
      • Morsch V.M.
      • Schetinger M.R.
      • Rue M.L.
      • Vogel F.S.
      • Lopes S.T.
      Influence of infection by Toxoplasma gondii on purine levels and E-ADA activity in the brain of mice experimentally infected mice.
      Moreover, the activity of ectoenzymes such as CD73 and CD39 cleave extracellular nucleotides, triggering the anti-inflammatory environment that minimizes microbicidal responses to T. gondii by microglia and astrocytes.
      • Mahamed D.A.
      • Toussaint L.E.
      • Bynoe M.S.
      CD73-generated adenosine is critical for immune regulation during Toxoplasma gondii infection.
      • Mahamed D.A.
      • Mills J.H.
      • Egan C.E.
      • Denkers E.Y.
      • Bynoe M.S.
      CD73-generated adenosine facilitates Toxoplasma gondii differentiation to long-lived tissue cysts in the central nervous system.
      The degradation of extracellular nucleotides in the central nervous system contributed to new young cyst formation and increased cyst numbers, suggesting the importance of P2 receptors and their pathway in the control of infection. In accordance with this result, the absence of the P2X7 receptor was characterized by the inability of ATP to activate the P2X7 receptors. This effect limited the proinflammatory innate immune response and favored the formation of young cysts, increasing morbidity and mortality as a result of parasite modulation of extracellular nucleotide concentrations. It also induced ectoenzyme activities, redirecting ATP to parasite metabolism, allowing parasite proliferation.
      In microglia and astrocytes, after activation of innate immune receptors such as NLR, Toll-like receptor, and P2 receptors, there was an immediate robust response with proinflammatory cytokines (IL-1β, IL-12, and IL-6) and chemokines (CCL2, CCL5, CXCL1, and CXCL10) that contributed to lymphocyte and myelocyte peripheral cell migration.
      • Silva N.M.
      • Manzan R.M.
      • Carneiro W.P.
      • Milanezi C.M.
      • Silva J.S.
      • Ferro E.A.
      • Mineo J.R.
      Toxoplasma gondii: the severity of toxoplasmic encephalitis in C57BL/6 mice is associated with increased ALCAM and VCAM-1 expression in the central nervous system and higher blood-brain barrier permeability.
      Interestingly, events associated with innate immune responses are crucial to control cerebral toxoplasmosis; the inflammatory monocyte Gr1+ promotes cellular responses to infection by IL-1β, IL-12, IL-2, IL-6, and ROS production inside the brain.
      • Biswas A.
      • Bruder D.
      • Wolf S.A.
      • Jeron A.
      • Mack M.
      • Heimesaat M.M.
      • Dunay I.R.
      Ly6C(high) monocytes control cerebral toxoplasmosis.
      In agreement with this result, increased levels of the same profile of cytokines and ROS production inside infected brains was observed. All of these mediators were absent or reduced in P2rx7−/− mice, suggesting that the P2X7 receptor is a key component in the promotion of innate immune responses involved in the control of cerebral toxoplasmosis.
      The balance between immune responses and parasite control in the brain is tightly regulated to protect the central nervous system from damage. Excessive production of inflammatory mediators and glial activation may induce severe neuroinflammatory processes that in turn may result in impaired synaptic plasticity, excitotoxicity, neuronal dysfunction, and death.
      • Parlog A.
      • Schluter D.
      • Dunay I.R.
      Toxoplasma gondii-induced neuronal alterations.
      • McCusker R.H.
      • Kelley K.W.
      Immune-neural connections: how the immune system's response to infectious agents influences behavior.
      Although the P2X7 receptor potentiates the production of inflammatory mediators in the brain, this mechanism appears to be crucial to the control of parasite replication, the formation of new cysts, and the spread of the parasite throughout the cerebral parenchyma. Therefore, these data support the notion that the P2X7 receptor mediates cerebral toxoplasmosis resistance through proinflammatory IL-1β, IL-12, and ROS production, independent of IFN-γ production in the brain, instead it is protective of generating neuropathologic inflammation and disease progression. Our results suggest that the mechanisms that limit P2X7 activation, including ectoenzymes, should be neutralized and/or P2X7 signaling should be stimulated and used as a therapeutic target to promote parasite control in the reactivation phase of cerebral toxoplasmosis.

      Acknowledgments

      We thank the funding agencies for the financial support and all authors who consistently contributed to this work.
      A.C.A.M.-S. designed and performed the experiments, analyzed the results, and wrote the manuscript; T.P.R., F.S., S.R.B.d.S., and V.R.F. performed the experiments and analyzed the results; L.E.B.S., C.M.T., and A.T.S.W. analyzed the results and revised the manuscript; and R.C.V. and R.C.-S. designed the experiments, analyzed the results, revised the manuscript, had full access to all the data in the study, and take responsibility for the integrity of the data and the accuracy of the data analysis, and are the guarantors of this work.

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