Produção Nacional
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Disruption of Purinergic Receptor P2X7 Signaling Increases Susceptibility to Cerebral Toxoplasmosis
2019; Elsevier BV; Volume: 189; Issue: 4 Linguagem: Inglês
10.1016/j.ajpath.2019.01.001
ISSN1525-2191
AutoresAline Cristina Abreu Moreira-Souza, Thuany Prado Rangel, Sthefani Rodrigues Batista da Silva, Vanessa Ribeiro Figliuolo, Luiz Eduardo Baggio Savio, Felipe Schmitz, Christina Maeda Takiya, Ângela Terezinha de Souza Wyse, Rossiane C. Vommaro, Robson Coutinho‐Silva,
Tópico(s)Heme Oxygenase-1 and Carbon Monoxide
ResumoToxoplasmosis 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 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.1Innes E.A. A brief history and overview of Toxoplasma gondii.Zoonoses Public Health. 2010; 57: 1-7Crossref PubMed Scopus (137) Google Scholar Toxoplasmosis affects one third of the world's population and infection by T. gondii can occur by ingestion of uncooked meat or contaminated water; the pathogen also can be acquired congenitally during pregnancy. In addition, immunocompromised organ recipients can reactivate old infections.2Montoya J.G. Liesenfeld O. 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The P2X7 receptor mediates Toxoplasma gondii control in macrophages through canonical NLRP3 inflammasome activation and ROS production.Front Immunol. 2017; 8: 1257Crossref PubMed Scopus (61) Google Scholar Polymorphisms in the human and murine P2X7-receptor gene generating less functional proteins have been associated directly with toxoplasmosis susceptibility in immunodeficient or immunocompetent hosts.29Jamieson 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.Genes Immun. 2010; 11: 374-383Crossref PubMed Scopus (78) Google Scholar, 30Sluyter R. Stokes L. 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Inflammatory early events associated to the role of P2X7 receptor in acute murine toxoplasmosis.Immunobiology. 2016; 222: 676-683Crossref PubMed Scopus (24) Google Scholar Considering that i) the infection by T. gondii parasite has no cure because there is no therapy with activity against the cyst form; ii) the manifestations of chronic toxoplasmosis irreversibly affects the central nervous system; and iii) the P2X7 receptor contributes to parasite control, the function of this receptor during cerebral toxoplasmosis was investigated using a chronic toxoplasmosis model in wild-type (WT) and P2rx7 knockout mice. P2rx7 deletion increased susceptibility to infection with a cystogenic T. gondii strain. P2rx7 knockout mice showed an increased number of brain cysts and low production of IL-12 and ROS. Moreover, these findings suggest that P2X7 is crucial for the promotion of an efficient immune response to protect the host against cerebral toxoplasmosis. 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. 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. 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. 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). 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.34LeBel C.P. Ischiropoulos H. Bondy S.C. Evaluation of the probe 2',7'-dichlorofluorescin as an indicator of reactive oxygen species formation and oxidative stress.Chem Res Toxicol. 1992; 5: 227-231Crossref PubMed Scopus (2231) Google Scholar 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.35Savio 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.Mol Neurobiol. 2017; 54: 6459-6470Crossref PubMed Scopus (33) Google Scholar 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.36Aebi H. Catalase in vitro.Methods Enzymol. 1984; 105: 121-126Crossref PubMed Scopus (18522) Google Scholar 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. 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). 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). 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). 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.37da Silva G.L. Sperotto N.D. Borges T.J. Bonorino C. Takyia C.M. Coutinho-Silva R. Campos M.M. Zanin R.F. Morrone F.B. P2X7 receptor is required for neutrophil accumulation in a mouse model of irritant contact dermatitis.Exp Dermatol. 2013; 22: 184-188Crossref PubMed Scopus (19) Google Scholar 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). 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). Because reactive nitrogen intermediates and ROS are associated with parasite death11Yarovinsky F. Innate immunity to Toxoplasma gondii infection.Nat Rev Immunol. 2014; 14: 109-121Crossref PubMed Scopus (277) Google Scholar and can be promoted by P2X7 activation during protozoal infections,23Chaves 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.Microbes Infect. 2009; 11: 842-849Crossref PubMed Scopus (66) Google Scholar, 25Correa 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.Microbes Infect. 2010; 12: 497-504Crossref PubMed Scopus (80) Google Scholar 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). 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. 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.4Skariah S. McIntyre M.K. Mordue D.G. Toxoplasma gondii: determinants of tachyzoite to bradyzoite conversion.Parasitol Res. 2010; 107: 253-260Crossref PubMed Scopus (97) Google Scholar 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 h
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