Produção Nacional
Revisado por pares
Toxoplasma gondii Prevents Neuron Degeneration by Interferon-γ-Activated Microglia in a Mechanism Involving Inhibition of Inducible Nitric Oxide Synthase and Transforming Growth Factor-β1 Production by Infected Microglia
2005; Elsevier BV; Volume: 167; Issue: 4 Linguagem: Inglês
10.1016/s0002-9440(10)61191-1
ISSN1525-2191
AutoresClaudia Rozenfeld, Rodrigo Martínez, Sérgio Henrique Seabra, Celso Sant’Anna, J. Gabriel R. Gonçalves, Marcelo T. Bozza, Vivaldo Moura‐Neto, Wanderley de Souza,
Tópico(s)Herpesvirus Infections and Treatments
ResumoInterferon (IFN)-γ, the main cytokine responsible for immunological defense against Toxoplasma gondii, is essential in all infected tissues, including the central nervous system. However, IFN-γ-activated microglia may cause tissue injury through production of toxic metabolites such as nitric oxide (NO), a potent inducer of central nervous system pathologies related to inflammatory neuronal disturbances. Despite potential NO toxicity, neurodegeneration is not commonly found during chronic T. gondii infection. In this study, we describe decreased NO production by IFN-γ-activated microglial cells infected by T. gondii. This effect involved strong inhibition of iNOS expression in IFN-γ-activated, infected microglia but not in uninfected neighboring cells. The inhibition of NO production and iNOS expression were parallel with recovery of neurite outgrowth when neurons were co-cultured with T. gondii-infected, IFN-γ-activated microglia. In the presence of transforming growth factor (TGF)-β1-neutralizing antibodies, the beneficial effect of the parasite on neurons was abrogated, and NO production reverted to levels similar to IFN-γ-activated uninfected co-cultures. In addition, we observed Smad-2 nuclear translocation, a hallmark of TGF-β1 downstream signaling, in infected microglial cultures, emphasizing an autocrine effect restricted to infected cells. Together, these data may explain a neuropreservation pattern observed during immunocompetent host infection that is dependent on T. gondii-triggered TGF-β1 secretion by infected microglia. Interferon (IFN)-γ, the main cytokine responsible for immunological defense against Toxoplasma gondii, is essential in all infected tissues, including the central nervous system. However, IFN-γ-activated microglia may cause tissue injury through production of toxic metabolites such as nitric oxide (NO), a potent inducer of central nervous system pathologies related to inflammatory neuronal disturbances. Despite potential NO toxicity, neurodegeneration is not commonly found during chronic T. gondii infection. In this study, we describe decreased NO production by IFN-γ-activated microglial cells infected by T. gondii. This effect involved strong inhibition of iNOS expression in IFN-γ-activated, infected microglia but not in uninfected neighboring cells. The inhibition of NO production and iNOS expression were parallel with recovery of neurite outgrowth when neurons were co-cultured with T. gondii-infected, IFN-γ-activated microglia. In the presence of transforming growth factor (TGF)-β1-neutralizing antibodies, the beneficial effect of the parasite on neurons was abrogated, and NO production reverted to levels similar to IFN-γ-activated uninfected co-cultures. In addition, we observed Smad-2 nuclear translocation, a hallmark of TGF-β1 downstream signaling, in infected microglial cultures, emphasizing an autocrine effect restricted to infected cells. Together, these data may explain a neuropreservation pattern observed during immunocompetent host infection that is dependent on T. gondii-triggered TGF-β1 secretion by infected microglia. 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Activation of Smads causes their translocation from cytoplasm to the nucleus where they control gene expression,36Heldin C-H Miyazono K ten Dijke P TGF-β signalling from cell membrane to nucleus through SMAD proteins.Nature. 1997; 390: 465-471Crossref PubMed Scopus (3301) Google Scholar, 37Wrana JL Attisano L The Smad pathway.Cytokine Growth Factor Rev. 2000; 11: 5-13Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar modulating several proteins including iNOS, which is involved with local inflammation.38Kim WK Hwang SY Oh ES Piao HZ Kim KW Han IO TGF-β1 repress activation and resultant death of microglia via inhibition of phosphatidylinositol 3-kinase activity.J Immunol. 2004; 172: 7015-7023PubMed Google Scholar, 39Lieb K Engels S Fiebich BL Inhibition of LPS-induced iNOS and NO synthesis in primary rat microglial cells.Neurochem Int. 2003; 42: 131-137Crossref PubMed Scopus (75) Google Scholar We have recently described an in vitro indirect neuron-protective effect of T. gondii infection, dependent on inhibition of NO production by activated microglial cells, which is indirectly regulated by infected astrocytes.40Rozenfeld C Martinez R Figueiredo RT Bozza MT Lima FR Pires AL Silva PM Bonomo A Lannes-Vieira J De Souza W Moura-Neto V Soluble factors released by Toxoplasma gondii-infected astrocytes down-modulate nitric oxide production by gamma interferon-activated microglia and prevent neuronal degeneration.Infect Immun. 2003; 71: 2047-2057Crossref PubMed Scopus (63) Google Scholar This phenomenon was shown to be mediated by PGE2 secretion from infected astrocytes followed by IL-10 production by IFN-γ-activated microglia.40Rozenfeld C Martinez R Figueiredo RT Bozza MT Lima FR Pires AL Silva PM Bonomo A Lannes-Vieira J De Souza W Moura-Neto V Soluble factors released by Toxoplasma gondii-infected astrocytes down-modulate nitric oxide production by gamma interferon-activated microglia and prevent neuronal degeneration.Infect Immun. 2003; 71: 2047-2057Crossref PubMed Scopus (63) Google Scholar Considering these data, the aim of the present study was to investigate a possible direct effect of T. gondii infection on IFN-γ-activated microglia cells that could favor neuron preservation, taking into consideration that, in addition to astrocytes, microglial cells are also able to harbor parasites.17Luder CG Giraldo-Velasquez M Sendtner M Gross U Toxoplasma gondii in primary rat CNS cells: differential contribution of neurons, astrocytes, and microglial cells for the intracerebral development and stage differentiation.Exp Parasitol. 1999; 93: 23-32Crossref PubMed Scopus (108) Google Scholar The observations here show that the inhibitory effect of the parasite on iNOS expression by IFN-γ-activated microglia seems to be dependent on TGF-β1 production by T. gondii-infected microglia, resulting in Smad-2 nucleus translocation, inhibition of NO production and neuron preservation. Chicken anti-TGF-β1-neutralizing antibody was obtained from R&D Systems. Mouse anti-human β-tubulin III antibody, aspirin (ASA), and murine recombinant IFN-γ were purchased from Sigma. Polyclonal antibody against iNOS, polyclonal antibody against Smad-2, and polyclonal antibody against TGF-β1 were obtained from Santa Cruz. The secondary antibodies used in this study were goat anti-rabbit fluorescein isothiocyanate-conjugated and goat anti-mouse Alexa, goat anti-mouse and goat anti-rabbit horseradish peroxidase-conjugated, which were purchased from Gibco BRL, and goat anti-rabbit cy3-conjugated from Sigma. Tachyzoites from the virulent RH strain of T. gondii were maintained within the intraperitoneal passages in Swiss Webster mice and were harvested for in vitro studies 2 days after infection. Mice were killed by CO2 inhalation and free tachyzoites were recovered from the peritoneal cavity after instilling 5 ml of Dulbecco's modified Eagle's medium (DMEM)/F12. The fluid obtained from infected mice was centrifuged at 200 × g for 7 minutes at room temperature to remove host cells and debris. The parasite-containing supernatant was collected and centrifuged at 1000 × g for 10 minutes. The pellet obtained was resuspended to a density of 106 parasites/ml in DMEM-F12. The parasites were then used within 30 to 40 minutes, and the viability was evaluated using a dye exclusion test with trypan blue. Chronically infected mice, orally infected with Pe strain (2 to 3 months), received a boost of RH tachizoites (104) 15 days before the blood punction and the obtaining of the serum by centrifugation. The serum of the control animal was also obtained but did not exhibit T. gondii staining in contrast to the serum of infected animals. In the tests using the titration specified in the paper, neither background nor nonspecific staining was observed on using the serum of infected animals. Murine astrocytes from BALB/c mice were cultured from the brain cortex of neonatal mice (age, between E-18 and P-0), following the procedure previously described,41Lima FSL Gervais A Colin C Izembart M Moura-Neto V Mallat M Regulation of microglial development: a novel role for thyroid hormone.J Neurosci. 2001; 21: 2028-2038Crossref PubMed Google Scholar with some modifications.40Rozenfeld C Martinez R Figueiredo RT Bozza MT Lima FR Pires AL Silva PM Bonomo A Lannes-Vieira J De Souza W Moura-Neto V Soluble factors released by Toxoplasma gondii-infected astrocytes down-modulate nitric oxide production by gamma interferon-activated microglia and prevent neuronal degeneration.Infect Immun. 2003; 71: 2047-2057Crossref PubMed Scopus (63) Google Scholar After 14 to 15 days, microglial cells were detached from the astrocyte monolayer by shaking the culture flasks for 30 minutes. The supernatants were collected and centrifuged, and the cells were reseeded on 24-well tissue culture chamber slides (Nunc, Inc.) with 5.5-mm diameter glass coverslips, at a final concentration of 5 × 105 cells/well in 500 μl of medium. After 40 minutes, the medium was replaced to remove nonadherent cells, and microglial cells were allowed to grow for an additional 24 to 48 hours before the experiments were started. Cells were found to be 98% microglia as judged by positive staining with isolectin b4 (peroxidase-labeled lectin from Bandeiraea simplicifolia BS-I, obtained from Sigma). After washing three times in serum-free DMEM/F12, microglial cells were allowed to interact with low parasite loads (10:1 and 1:5, host cell:parasite ratio) for 2 hours. To eliminate free parasites, after this period, cultures were washed three times with DMEM/F-12. The cells were then activated with IFN-γ (500 U/ml) in a final volume of 500 U/well, for a period of 18 to 24 hours. No cell lysis was observed in this period. Supernatants were tested for IL-10 using a murine sandwich enzyme-linked immunosorbent assay kit (Pharmingen, La Jolla, CA) according to the manufacturer's instructions. Apoptotic cells were detected by TUNEL assay using APO-BrdU TUNEL assay kit (A-23210; Molecular Probes, Eugene, OR) according to the manufacturer's instructions. Primary dissociated cortical neurons were prepared as previously described42Gomes FC Maia CG de Menezes JR Neto VM Cerebellar astrocytes treated by thyroid hormone modulate neuronal proliferation.Glia. 1999; 25: 247-255Crossref PubMed Scopus (82) Google Scholar with some modifications. Briefly, timed pregnancy mice were sacrificed on the 17th or 18th gestational day, and embryos were removed by caesarian section. After cortex dissection as described above for glial cultures, cells were dissociated in DMEM/F-12 medium supplemented with 10% fetal bovine serum. Neurons (105/well) were then plated on top of microglial monolayers, previously infected or not, 2 hours before, as described above. After 1 hour of seeding neurons, the medium was carefully replaced (500 μl/well) and then IFN-γ (500 U/ml) was added in the absence or in the presence of TGF-β1-neutralizing antibodies (20 ng/ml) or ASA (100 μmol/L) for 24 hours. As previously described,40Rozenfeld C Martinez R Figueiredo RT Bozza MT Lima FR Pires AL Silva PM Bonomo A Lannes-Vieira J De Souza W Moura-Neto V Soluble factors released by Toxoplasma gondii-infected astrocytes down-modulate nitric oxide production by gamma interferon-activated microglia and prevent neuronal degeneration.Infect Immun. 2003; 71: 2047-2057Crossref PubMed Scopus (63) Google Scholar cultured cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS), permeabilized with 0.2% Triton X-100, and endogenous peroxidase activity was abolished with 3% H2O2. Cells were incubated with 5% bovine serum albumin (Gibco BRL) in PBS (blocking solution) and subsequently with the specified mouse anti-human β-tubulin III antibody (1:400 dilution, Sigma), diluted in blocking solution. Then, the cells were incubated with horseradish peroxidase-conjugated goat anti-mouse immunoglobulin (1:200 dilution) and peroxidase activity was revealed using a diaminobenzidine peroxidase substrate kit, which stained the cells black (Vector Laboratories). After this first step, in experiments of double staining, incubation with anti-iNOS polyclonal antibody was performed (1:100), followed by incubation with secondary horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin (1:200 dilution). Secondary revelation was performed using the VIP (purple) substrate kit (Vector Laboratories). All antibodies were diluted in blocking solution. The preparations were dried in air and coverslips were mounted in Entellan (Merck). Cultured cells were fixed with 4% paraformaldehyde and 4% sucrose in phosphate-buffered saline (PBS) for 20 minutes and permeabilized with 0.2% Triton X-100 for 5 minutes at room temperature. Cells were incubated with 5% bovine serum albumin (Gibco BRL) plus inactivated normal mice serum (1:100) in PBS (blocking solution) for 30 minutes. Subsequently cells were incubated with anti-iNOS polyclonal antibody for 1 hour, followed by incubation with anti-rabbit fluorescein isothiocyanate-stained antibody (1:500). T. gondii tachyzoites were stained using serum (1:100) of chronically infected mice (Pe strain) boosted 1 week before bleeding with parasites of the same strain. After incubation for 1 hour at room temperature, a secondary goat anti-mouse rhodamine-stained antibody (1:500) was used. The same procedures, with minor changes, were used with Smad-2 immunostaining (1:50). The incubation was performed overnight at 4°C, followed by secondary anti-rabbit Cy3 stained antibody (1:5000) incubation for 1 hour. All antibodies were diluted in blocking solution. The preparations were washed several times in PBS between all steps and then coverslips were mounted in N-propyl-gallate solution. Supernatants from microglial cells and neuron-microglia co-cultures were assayed for nitrite content, which reflects NO production, using Griess reagent (0.1% naphthylethylene diamine dihydrochloride and 1% sulfanilamide plus 2.5% phosphoric acid in equal volumes) as described previously.43Ding AH Nathan CF Stuehr DJ Release of reactive nitrogen intermediates and reactive oxygen intermediates from mouse peritoneal macrophages. Comparison of activating cytokines and evidence for independent production.J Immunol. 1988; 141: 2407-2412PubMed Google Scholar Neurons stained for β-tubulin III were photographed in a Nikon microscope. Photos were scanned and neurite length was analyzed using the Sigma Scan Pro Software (Jandel Scientific). Three independent experiments were performed and at least 100 neurons were counted per sample in six or seven randomly chosen fields. Data were analyzed by Student's t-test or analysis of variance. Probability values (P) of 0.05 or less were considered significant. An inhibitory effect of the T. gondii on NO production by IFN-γ-activated microglia mediated indirectly by soluble factors released by infected astrocytes was recently described.40Rozenfeld C Martinez R Figueiredo RT Bozza MT Lima FR Pires AL Silva PM Bonomo A Lannes-Vieira J De Souza W Moura-Neto V Soluble factors released by Toxoplasma gondii-infected astrocytes down-modulate nitric oxide production by gamma interferon-activated microglia and prevent neuronal degeneration.Infect Immun. 2003; 71: 2047-2057Crossref PubMed Scopus (63) Google Scholar Microglia, besides astrocytes, are also a target of the parasite during CNS infection, and so the direct effect of the T. gondii on NO produced by microglial cells was tested. As shown in Figure 1, treatment of microglial cells with IFN-γ for 16 to 18 hours induced a strong NO production as attested by measurement of nitrite accumulation in the supernatant. A suppressive effect of the parasite on NO production by IFN-γ-activated microglia was clearly observed even in parasite loads as low as 5:1, host cell:parasite ratio (P < 0.05). This NO inhibitory effect was higher using increased parasite load infection (P < 0.01), reaching control levels, without apparent cell lysis detection. This result suggests that the inhibition of NO production was directly correlated with the parasite:host cell ratio used. Although NO synthesis by IFN-γ-activated microglia is mainly dependent on iNOS using l-arginine as precursor, nonenzymatic production may also occur. In addition some pathogens may lead to a reduction of nitrite levels by acting as scavengers or consuming it44Trajkovic V Stepanovic S Samardzic T Jankovic V Badovinac V Mostarica Stojkovic M Cryptococcus neoformans neutralizes macrophage and astrocyte derived nitric oxide without interfering with inducible nitric oxide synthase induction or catalytic activity—possible involvement of nitric oxide consumption.Scand J Immunol. 2000; 51: 384-391Crossref PubMed Scopus (12) Google Scholar and consequently altering the bioavailability of NO. To investigate if the reduction of nitrite levels detected after T. gondii infection was correlated with a down-modulation of iNOS expression, immunofluorescence microscopy localization of iNOS was performed. Microglial cells in the absence of stimulation, showed undetectable basal levels of iNOS expression (Figure 1B), in contrast to IFN-γ-activated microglia where the enzyme was overexpressed (Figure 1C). This observation is in agreement with NO detection in cell supernatants (Figure 1A), strongly suggesting an enzymatic source of NO production after IFN-γ microglia activation. In the presence of a high T. gondii:host cell ratio 5:1 of infection, the expression of iNOS by IFN-γ-activated microglia was practically undetectable (Figure 1D). In the same manner, T. gondii-infected microglial cells in the absence of IFN-γ expressed
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