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Intraphagosomal Peroxynitrite as a Macrophage-derived Cytotoxin against Internalized Trypanosoma cruzi

CONSEQUENCES FOR OXIDATIVE KILLING AND ROLE OF MICROBIAL PEROXIREDOXINS IN INFECTIVITY*
  • María Noel Alvarez
    Affiliations
    Departamento de Bioquímica and Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la República, Montevideo 11800, Uruguay
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  • Gonzalo Peluffo
    Affiliations
    Departamento de Bioquímica and Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la República, Montevideo 11800, Uruguay
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  • Lucía Piacenza
    Affiliations
    Departamento de Bioquímica and Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la República, Montevideo 11800, Uruguay
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  • Rafael Radi
    Correspondence
    A Howard Hughes International Research Scholar. To whom correspondence should be addressed: Dpto. de Bioquímica, Facultad de Medicina, Universidad de la República, Avda. General Flores 2125, Montevideo 11800, Uruguay. Tel.: 5982-9249561; Fax: 5982-9249563;
    Affiliations
    Departamento de Bioquímica and Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la República, Montevideo 11800, Uruguay
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  • Author Footnotes
    * This work was supported by grants from the Howard Hughes Medical Institute and the International Centre for Genetic Engineering and Biotechnology (to R. R.), the Fondo Clemente Estable (Programa de Desarrollo Tecnológico) (to L. P.), and the Comisión Sectorial de Investigación Científica, Universidad de la República (to M. N. A.).
    The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1 and 2.
Open AccessPublished:November 23, 2010DOI:https://doi.org/10.1074/jbc.M110.167247
      Macrophage-derived radicals generated by the NADPH oxidase complex and inducible nitric-oxide synthase (iNOS) participate in cytotoxic mechanisms against microorganisms. Nitric oxide (NO) plays a central role in the control of acute infection by Trypanosoma cruzi, the causative agent of Chagas disease, and we have proposed that much of its action relies on macrophage-derived peroxynitrite (ONOO + ONOOH) formation, a strong oxidant arising from the reaction of NO with superoxide radical (O2̇̄). Herein, we have shown that internalization of T. cruzi trypomastigotes by macrophages triggers the assembly of the NADPH oxidase complex to yield O2̇̄ during a 60–90-min period. This does not interfere with IFN-γ-dependent iNOS induction and a sustained NO production (∼24 h). The major mechanism for infection control via reactive species formation occurred when NO and O2̇̄ were produced simultaneously, generating intraphagosomal peroxynitrite levels compatible with microbial killing. Moreover, biochemical and ultrastructural analysis confirmed cellular oxidative damage and morphological disruption in internalized parasites. Overexpression of cytosolic tryparedoxin peroxidase in T. cruzi neutralized macrophage-derived peroxynitrite-dependent cytotoxicity to parasites and favored the infection in an animal model. Collectively, the data provide, for the first time, direct support for the action of peroxynitrite as an intraphagosomal cytotoxin against pathogens and the premise that microbial peroxiredoxins facilitate infectivity via decomposition of macrophage-derived peroxynitrite.

      Introduction

      Macrophages represent one of the first lines of defense against invading intracellular pathogens through their ability to recognize, ingest, and destroy microorganisms. The detection of infectious agents by pattern recognition receptors initiates signal transduction pathways, which leads to activation of the membrane-bound NADPH oxidase and induction of nitric-oxide synthase 2 (iNOS),
      The abbreviations used are iNOS, inducible nitric-oxide synthase; NO, nitric oxide; O2̇̄, superoxide radical; OH, hydroxyl radical; NO2, nitrogen dioxide radical; CO3̇̄, carbonate radical; DHR, dihydrorhodamine; NBT, nitro blue tetrazolium; DAF-2DA, 4,5-diaminofluorescein diacetate; DPI, diphenyliodonium; l-NMMA, NG-monomethyl-l-arginine; TcCPX, T. cruzi cytosolic tryparedoxin peroxidase; MPO, myeloperoxidase; DMPO, 5,5-dimethylpyrroline-N-oxide; SOD, superoxide dismutase; dPBS, Dulbecco's PBS; DIC, differential interference contrast; RH 123, rhodamine 123.
      central components of oxidative-killing mechanisms. The NADPH oxidase complex is inactive in resting phagocytes and assembles upon stimulation, resulting in formation of antimicrobial superoxide radicals (O2̇̄) within the phagosome (
      • Babior B.M.
      ,
      • Groemping Y.
      • Rittinger K.
      ). The univalent reduction of oxygen by NADPH oxidase occurs immediately after phagocytosis, yielding high rates of O2̇̄ for at least 90 min (
      • Alvarez M.N.
      • Trujillo M.
      • Radi R.
      ). Superoxide radical production can also result in the formation of H2O2 in the phagocytic vacuole either by spontaneous or extracellular SOD-catalyzed dismutation (
      • De Groote M.A.
      • Ochsner U.A.
      • Shiloh M.U.
      • Nathan C.
      • McCord J.M.
      • Dinauer M.C.
      • Libby S.J.
      • Vazquez-Torres A.
      • Xu Y.
      • Fang F.C.
      ,
      • Fridovich I.
      ). Macrophage cytotoxicity is enhanced by the presence of proinflammatory cytokines such as IFN-γ, which are secreted by natural killer cells (NK) in the first instance and by CD4+ T helper-1 (Th1) cells once a specific immune response against the pathogen is established (
      • Aliberti J.C.
      • Cardoso M.A.
      • Martins G.A.
      • Gazzinelli R.T.
      • Vieira L.Q.
      • Silva J.S.
      ,
      • Cardillo F.
      • Voltarelli J.C.
      • Reed S.G.
      • Silva J.S.
      ). Following cytokine activation, macrophages produce large amounts of nitric oxide (NO) because of induction of iNOS (
      • Xie Q.W.
      • Whisnant R.
      • Nathan C.
      ,
      • Martin E.
      • Nathan C.
      • Xie Q.W.
      ,
      • MacMicking J.
      • Xie Q.W.
      • Nathan C.
      ). In general terms, neither O2̇̄ nor NO has been shown to be particularly cytotoxic per se, although the concomitant production of both radicals potentiates cytotoxicity (
      • Ischiropoulos H.
      • Zhu L.
      • Beckman J.S.
      ,
      • Nathan C.
      • Shiloh M.U.
      ,
      • Alvarez M.N.
      • Piacenza L.
      • Irigoín F.
      • Peluffo G.
      • Radi R.
      ).
      We and others have proposed that the concerted actions of phagosomal NADPH oxidase and iNOS leads to the formation of peroxynitrite,
      The term peroxynitrite refers to the sum of peroxynitrite anion (ONOO) and peroxynitrous acid (ONOOH), pKa = 6.8 (
      • Radi R.
      • Peluffo G.
      • Alvarez M.N.
      • Naviliat M.
      • Cayota A.
      ,
      • Ferrer-Sueta G.
      • Radi R.
      ).
      a potentially potent microbicidal species inside the phagosome (
      • Ischiropoulos H.
      • Zhu L.
      • Beckman J.S.
      ,
      • Nathan C.
      • Shiloh M.U.
      ,
      • Denicola A.
      • Rubbo H.
      • Rodríguez D.
      • Radi R.
      ,
      • Evans T.J.
      • Buttery L.D.
      • Carpenter A.
      • Springall D.R.
      • Polak J.M.
      • Cohen J.
      ,
      • Vazquez-Torres A.
      • Jones-Carson J.
      • Mastroeni P.
      • Ischiropoulos H.
      • Fang F.C.
      ,
      • Hickman-Davis J.
      • Gibbs-Erwin J.
      • Lindsey J.R.
      • Matalon S.
      ). Because of its negative charge, the O2̇̄ radical (pKa = 4.8) remains within this organelle, whereas NO, being a hydrophobic radical, diffuses from the cytosol or phagosomal membrane-associated iNOS (
      • Vodovotz Y.
      • Russell D.
      • Xie Q.W.
      • Bogdan C.
      • Nathan C.
      ) into the phagocytic vacuole. The reaction between NO and O2̇̄ yields peroxynitrite (
      • Nauser T.
      • Koppenol W.
      ,
      • Radi R.
      • Peluffo G.
      • Alvarez M.N.
      • Naviliat M.
      • Cayota A.
      ), a strong one- and two-electron oxidant and a precursor of secondary species including hydroxyl (OH), nitrogen dioxide (NO2), and carbonate (CO3̇̄) radicals (
      • Ferrer-Sueta G.
      • Radi R.
      ). Peroxynitrite and peroxynitrite-derived radicals are capable of reacting with different biological targets including protein amino acid residues (e.g. thiol oxidation (
      • Quijano C.
      • Alvarez B.
      • Gatti R.M.
      • Augusto O.
      • Radi R.
      ), tyrosine nitration (
      • Radi R.
      )) transition metal-containing centers (
      • Romero N.
      • Radi R.
      • Linares E.
      • Augusto O.
      • Detweiler C.D.
      • Mason R.P.
      • Denicola A.
      ), lipids (
      • Rubbo H.
      • Radi R.
      • Trujillo M.
      • Telleri R.
      • Kalyanaraman B.
      • Barnes S.
      • Kirk M.
      • Freeman B.A.
      ), and nucleic acids (
      • Szabó C.
      ), reactions that can ultimately promote cell death by either apoptotic or necrotic pathways (
      • Szabó C.
      • Ischiropoulos H.
      • Radi R.
      ). The role of various oxidative mechanisms in the microbicidal activity of macrophages depends on a number of factors including, in the case of the oxidative mechanisms, a dynamic balance between the activation state of macrophages and the sensitivity of targets cells to oxidants (
      • Nathan C.
      • Shiloh M.U.
      ,
      • Piacenza L.
      • Peluffo G.
      • Alvarez M.N.
      • Kelly J.M.
      • Wilkinson S.R.
      • Radi R.
      ). However, the actual formation of peroxynitrite in the phagosome at levels that could be cytotoxic to invading microorganisms has remained elusive. Although in simple biochemical systems the reaction of NO with O2̇̄ is very efficient, approaching the diffusion-controlled limit (
      • Ferrer-Sueta G.
      • Radi R.
      ), in biological systems, competition reactions (e.g. SOD-catalyzed O2̇̄ dismutation) (
      • De Groote M.A.
      • Ochsner U.A.
      • Shiloh M.U.
      • Nathan C.
      • McCord J.M.
      • Dinauer M.C.
      • Libby S.J.
      • Vazquez-Torres A.
      • Xu Y.
      • Fang F.C.
      ), rapid diffusion of NO across biomembranes (
      • Quijano C.
      • Romero N.
      • Radi R.
      ), and temporal mismatch between NO and O2̇̄ production (
      • Eu J.P.
      • Liu L.
      • Zeng M.
      • Stamler J.S.
      ,
      • Pfeiffer S.
      • Lass A.
      • Schmidt K.
      • Mayer B.
      ,
      • Palazzolo-Ballance A.M.
      • Suquet C.
      • Hurst J.K.
      ), among other factors, can preclude peroxynitrite formation. In addition, once formed, much of the peroxynitrite may be decomposed by reacting with noncritical targets and antioxidant enzymes (i.e. peroxiredoxins) (reviewed in Ref.
      • Ferrer-Sueta G.
      • Radi R.
      ), and therefore its potential cytotoxic effects are attenuated or even fully neutralized.
      Trypanosoma cruzi is an obligate intracellular pathogen responsible for Chagas disease. It has been estimated that 8 million people are currently infected by the parasite, with 20% of Latin America's population (109 million) at risk resulting in 41,000 new cases each year (
      • Rassi Jr., A.
      • Rassi A.
      • Marin-Neto J.A.
      ). Moreover, the disease is spreading worldwide as a result of migration (mammalian hosts and insect vectors), HIV co-infection, blood transfusion, and organ transplantation. During its life cycle, T. cruzi undergoes extensive morphological and biochemical changes; non-infective epimastigotes proliferate in the gut of the insect vector where they differentiate into the infective metacyclic trypomastigotes. When initially infecting a host, the metacyclic form invades mainly host macrophages, where they can be destroyed by host immune mechanisms, including phagocytosis, or transformed into replicative intracellular amastigotes. After several cycles of binary division, host cell disruption occurs, and infective forms access the bloodstream and are able to invade cells from any tissue, such as the myocardium, smooth muscle, and central nervous system.
      Biologically relevant levels of peroxynitrite are cytotoxic to non- infective epimastigote parasites (
      • Denicola A.
      • Rubbo H.
      • Rodríguez D.
      • Radi R.
      ). In vitro experiments have revealed that peroxynitrite addition to T. cruzi epimastigotes results in severe alterations of energy charge, calcium homeostasis, and depletion of trypanothione, the main low molecular weight thiol found in trypanosomatids (
      • Thomson L.
      • Denicola A.
      • Radi R.
      ). We have shown that macrophage-derived peroxynitrite has the capacity to diffuse and exert cytotoxicity against epimastigotes in co-culture experiments (
      • Alvarez M.N.
      • Piacenza L.
      • Irigoín F.
      • Peluffo G.
      • Radi R.
      ). Additionally, parasites overexpressing peroxiredoxins, antioxidant enzymes known to readily detoxify peroxynitrite (
      • Trujillo M.
      • Budde H.
      • Piñeyro M.D.
      • Stehr M.
      • Robello C.
      • Flohé L.
      • Radi R.
      ,
      • Chen L.
      • Xie Q.W.
      • Nathan C.
      ) and recently revealed as virulent factors in the human infection (
      • Piacenza L.
      • Zago M.P.
      • Peluffo G.
      • Alvarez M.N.
      • Basombrio M.A.
      • Radi R.
      ), are resistant to this reactive species (
      • Piacenza L.
      • Peluffo G.
      • Alvarez M.N.
      • Kelly J.M.
      • Wilkinson S.R.
      • Radi R.
      ). As early as 1974, the central role of macrophage phagocytosis in the control of T. cruzi infection was determined (
      • Kierszenbaum F.
      • Knecht E.
      • Budzko D.B.
      • Pizzimenti M.C.
      ). Later, the importance of macrophages in in vivo infection was evidenced by experiments in which resistance to T. cruzi was associated with the production of IFN-γ and IL-12 in animal models (
      • Torrico F.
      • Heremans H.
      • Rivera M.T.
      • Van Marck E.
      • Billiau A.
      • Carlier Y.
      ,
      • Oliveira M.A.
      • Santiago H.C.
      • Lisboa C.R.
      • Ceravollo I.P.
      • Trinchieri G.
      • Gazzinelli R.T.
      • Vieira L.Q.
      ). Moreover, early treatment with IFN-γ was able to reverse the course of infection (
      • McCabe R.E.
      • Mullins B.T.
      ,
      • Wirth J.J.
      • Kierszenbaum F.
      ,
      • Reed S.G.
      • Nathan C.F.
      • Pihl D.L.
      • Rodricks P.
      • Shanebeck K.
      • Conlon P.J.
      • Grabstein K.H.
      ), presumably by controlling the early replication of parasites in host macrophages, with the participation of NO (
      • Muñoz-Fernández M.A.
      • Fernández M.A.
      • Fresno M.
      ,
      • Roman-Campos D.
      • Duarte H.L.
      • Sales Jr., P.A.
      • Natali A.J.
      • Ropert C.
      • Gazzinelli R.T.
      • Cruz J.S.
      ).
      Macrophage-derived O2̇̄ production triggered by T. cruzi is a matter of debate, with discussion centered on the ability of trypomastigotes to activate NADPH oxidase (
      • McCabe R.E.
      • Mullins B.T.
      ,
      • Cardoni R.L.
      • Antunez M.I.
      • Morales C.
      • Nantes I.R.
      ,
      • Tanaka Y.
      • Tanowitz H.
      • Bloom B.R.
      ). In regard to the modulation of NO production, contradictory results have been reported; although a negative regulation of iNOS induction by epimastigotes has been proposed (
      • Stempin C.
      • Giordanengo L.
      • Gea S.
      • Cerbán F.
      ,
      • Stempin C.C.
      • Garrido V.V.
      • Dulgerian L.R.
      • Cerbán F.M.
      ), experiments performed with trypomastigotes have shown potentiation of cytokine-mediated iNOS induction (
      • Bergeron M.
      • Olivier M.
      ). Thus, the occurrence of O2̇̄, NO, and their reaction product, peroxynitrite, and how these reactive species participate independently or synergically in the ability of host macrophages to control T. cruzi infection require an unambiguous analysis. In this work, we first studied whether T. cruzi infection of activated macrophages results in the simultaneous production of NO and O2̇̄ and leads to the intraphagosomal formation of peroxynitrite. We then evaluated the capacity of peroxynitrite versus that of NO and O2̇̄ (and H2O2) alone to efficiently serve as cytotoxins to control the load of internalized parasites. Finally, we tested the role of the cytosolic parasite peroxiredoxin in neutralizing the effects of host cell-derived oxidants in vitro and in vivo with the possible consequent promotion of infectivity. The data presented herein assist in understanding the mechanisms of oxidant-dependent killing of internalized pathogens.

      EXPERIMENTAL PROCEDURES

      Reagents

      Murine recombinant IFN-γ, human myeloperoxidase (MPO), and the anti-human MPO antibody were purchased from Calbiochem. Dulbecco's modified Eagle's medium (DMEM), lipopolysaccharide (LPS), luminol, NG-monomethyl-l-arginine (l-NMMA), apocynin, [5,6-3H]uridine (30 Ci mmol−1), Geneticin (G418), nitro blue tetrazolium (NBT), diphenyliodonium (DPI), and the anti-N-terminal region of actin antibody were from Sigma. Lab-Tek tissue culture chamber slides were from Nunc. 4′-6-Diamidino-2-phenylindole (DAPI), dihydrorhodamine (DHR), and Alexa-Fluor 488- and 594-conjugated anti-rabbit antibodies were from Invitrogen. 4,5-Diaminofluorescein diacetate was from Alexis Biochemicals. All other reagents were of research grade quality.

      Parasite Culture and Differentiation

      T. cruzi epimastigotes (CL-Brener) were cultured at 28 °C in brain-heart infusion medium as described previously (
      • Piacenza L.
      • Peluffo G.
      • Radi R.
      ). Parasites overexpressing cytosolic tryparedoxin peroxidase (TcCPX) were kindly provided by Dr. Shane Wilkinson (Queen Mary University of London, United Kingdom) (
      • Wilkinson S.R.
      • Temperton N.J.
      • Mondragon A.
      • Kelly J.M.
      ) and were cultured in brain-heart infusion medium containing 250 μg ml−1 G418 (Sigma) (
      • Piacenza L.
      • Peluffo G.
      • Alvarez M.N.
      • Kelly J.M.
      • Wilkinson S.R.
      • Radi R.
      ). Parasites were differentiated into the infective metacyclic stage under chemically defined conditions as described previously (
      • Contreras V.T.
      • Salles J.M.
      • Thomas N.
      • Morel C.M.
      • Goldenberg S.
      ). Briefly, epimastigotes were collected by centrifugation at 800 × g for 10 min at 25 °C, washed three times in 10 ml of triatomine artificial urine medium (TAU: 190 mm NaCl, 17 mm KCl, 2 mm MgCl2, 2 mm CaCl2, 8 mm phosphate, pH 6, and 0.035% sodium bicarbonate), and resuspended at a cell density of 3–5 × 108 cells ml−1. After 2 h of incubation at 28 °C, parasites were diluted in TAU-3AAG medium (TAU, pH 6, supplemented with three amino acids: 10 mm l-proline, 50 mm sodium l-glutamate, 2 mm sodium l-aspartate, and 10 mm glucose) at 3–5 × 106 cells ml−1. Cells were incubated for 96 h at 28 °C in 75-cm2 culture flasks in a horizontal position to allow adhesion of epimastigotes to the flask surface, an important step in enabling parasites to be transformed into the infective metacyclic trypomastigote stage (
      • Bonaldo M.C.
      • Souto-Padron T.
      • de Souza W.
      • Goldenberg S.
      ).

      Macrophages Culture

      The murine macrophage cell line J774A.1 (American Type Culture Collection (ATCC-TIB-67)) was cultured in DMEM (Sigma) supplemented with l-glutamine (2 mm), penicillin (100 units ml−1), streptomycin (100 mg l−1), and 10% heat-inactivated fetal bovine serum at 37 °C in a 5% CO2 atmosphere.

      Macrophage O2̇̄ Production

      Luminol Chemiluminescence

      Macrophages were cultured in 24-well plates, and O2̇̄ production was assayed by chemiluminescence studies as described previously (
      • Alvarez M.N.
      • Piacenza L.
      • Irigoín F.
      • Peluffo G.
      • Radi R.
      ). Briefly, macrophages were incubated under different experimental conditions in Dulbecco's PBS (dPBS), pH 7.3, containing 5 mm glucose, 1 mm l-arginine, and 100 μm luminol; the time course of luminol chemiluminescence was followed for 100 min in a luminescence plate reader at 37 °C (Lumistar, BMG Labtechnologies).

      Intracellular Detection of Reduced NBT

      Macrophages were seeded in Lab-Tek tissue culture chamber slides (Nunc) and infected with T. cruzi metacyclic trypomastigotes (5:1, parasite:macrophage ratio) in dPBS containing 1 mg/ml NBT (
      • Baehner R.L.
      • Boxer L.A.
      • Davis J.
      ). Slides were incubated at 37 °C for 30 min, rinsed in warmed dPBS, fixed for 10 min in 4% (v/v) formaldehyde in PBS at room temperature, and stained with DAPI (5 μg ml−1). Cells that contained formazan-stained inclusions within phagocytic vacuoles were observed by differential interference contrast (DIC) microscopy, and internalized trypomastigotes were evidenced by colocalization of the formazan deposits with DAPI fluorescence.

      Oxygen Consumption by Macrophages

      Macrophage mitochondrial respiration and respiratory burst were evaluated using a high resolution electrode, Oxygraph 2K (Oroboros Instruments, Innsbruck, Austria). Cells were cultured in 6-well plates and challenged with opsonised zymosan (
      • Baxter M.A.
      • Leslie R.G.
      • Reeves W.G.
      ) or T. cruzi metacyclic trypomastigotes in DMEM. Interaction was synchronized by plate centrifugation (800 × g, 5 min), and following a 15-min incubation at 37 °C non-internalized zymosan or parasites were removed. Macrophages were resuspended at 1 × 106 cells/ml in DMEM, and O2 consumption was recorded at 37 °C. The rate of oxygen consumption was calculated using DatLab 4.3 software (Oroboros Instruments) and was expressed as nmol O2 min−1/106 cells. Oxygen consumption inhibited by 1 mm potassium cyanide corresponds to mitochondrial respiration. The cyanide-insensitive fraction was inhibited by 100 μm DPI and corresponds to NADPH oxidase activity (
      • Hancock J.T.
      • Jones O.T.
      ).

      Macrophage NO Production

      The induction of the macrophage iNOS was triggered by incubating the cells with 200–300 units ml−1 IFN-γ (Calbiochem) plus 3 μg ml−1 LPS (Sigma) for 5 h (
      • Alvarez M.N.
      • Trujillo M.
      • Radi R.
      ). NO production by unstimulated or IFN-γ/LPS-activated macrophages was evaluated in tissue culture. Cells were seeded at 1 × 106 cells/well in the presence or absence of T. cruzi epimastigotes or chemically differentiated metacyclic trypomastigotes (see above). Following incubation (for 24 h), supernatants were collected, and the concentration of nitrite (NO2) was determined spectrophotometrically at 540 nm using the Griess method with NaNO2 as the standard (
      • Alvarez M.N.
      • Trujillo M.
      • Radi R.
      ,
      • Granger D.L.
      • Taintor R.R.
      • Boockvar K.S.
      • Hibbs Jr., J.B.
      ). Macrophage-NO production was also evaluated using the cell-permeable fluorescence probe 4,5-diaminofluorescein diacetate (DAF-2DA) (
      • Nakatsubo N.
      • Kojima H.
      • Kikuchi K.
      • Nagoshi H.
      • Hirata Y.
      • Maeda D.
      • Imai Y.
      • Irimura T.
      • Nagano T.
      ). After 5 h of incubation with the iNOS inducers, the medium was replaced with dPBS with 5 mm glucose and 1 mm l-arginine containing 5 μm DAF-2DA. Probe oxidation was followed in a fluorescence plate reader at 37 °C (Fluostar, BMG Labtechnologies) with filters at λex = 485 nm and λem = 520 nm. Under our experimental conditions, the peroxynitrite-dependent reactions (
      • Roychowdhury S.
      • Luthe A.
      • Keilhoff G.
      • Wolf G.
      • Horn T.F.
      ) did not interfere with the fluorophore.

      Measurements of iNOS Messenger RNA Levels

      Macrophages were seeded in 6-well plates (5 × 106 cells/well) and infected at a 10:1 parasite:macrophage ratio. After 3 h of infection total RNA was extracted using the PureLinkTM Micro-to-MidiTM Total RNA purification system (Invitrogen), and a cDNA copy was obtained using the SuperScriptTM III first-strand system for RT-PCR (Invitrogen). iNOS mRNA in each condition was amplified using the primers 5′-CTAAGAGTCACCAAAATGGCTCCC-3′ and 5′-ACCAGAGGCAGCACATCAAAGC-3′, which yielded an 875-bp band of murine iNOS. The housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified to verify equal cDNA loading in each condition with the primers 5′-CTGAGAACGGGAAGCTTGTC-3′ and 5′-CCTGCTTCACCACCTTCTTG-3′, which yielded a 600-bp band. Polymerase chain reaction was carried out using 5 μg of cDNA at an annealing temperature of 58 °C. PCR products were resolved by electrophoresis in 2% agarose gel, fragments were visualized with ethidium bromide, and gels were registered with a digital Polaroid camera.

      Immunochemical Evaluation of Myeloperoxidase

      Mice neutrophils, used as a positive control for MPO detection, were purified from peripheral blood as described (
      • Luo Y.
      • Dorf M.E.
      ). For protein extraction, neutrophils and J774A-1 macrophages were lysed in radioimmune precipitation assay buffer (150 mm NaCl, 10 mm Na2HPO4, pH 7.2, 0.1% SDS, 1% Triton X-100, and 1% sodium deoxycholate) supplemented with a mixture of protease inhibitors (Sigma). For immunoblotting, samples were separated by SDS-PAGE (10%) and transferred onto a PVDF membrane (Millipore). The membrane was incubated with a rabbit polyclonal anti-human MPO antibody (Calbiochem) and horseradish peroxidase-conjugated antibody as the secondary antibody. The blots were developed with ECL reagent (Amersham Biosciences). The nitrocellulose membrane was stripped and reblotted with anti-actin antibody (Sigma).

      Intracellular Dihydrorhodamine Oxidation

      Metacyclic trypomastigotes were incubated for 20 min in dPBS, pH 7.4, containing 50 μm DHR for intracellular probe incorporation and washed twice in dPBS before the assay. Macrophages were infected with DHR-loaded metacyclic trypomastigotes for 30 min at 37 °C and washed in dPBS to remove non-internalized parasites. Intracellular rhodamine 123 (RH 123, oxidation product of DHR) formation under the different macrophage stimulation conditions was measured in a fluorescence plate reader at 37 °C (Fluostar, BMG Labtechnologies) with filters at λex = 485 nm and λem = 520 nm (
      • Alvarez M.N.
      • Trujillo M.
      • Radi R.
      ). To evaluate rhodamine localization, cells were examined by fluorescence microscopy, and digital photographs of infected macrophages were recorded.

      Immunocytochemistry Studies

      Macrophages cultured in Lab-Tek glass chamber slides were exposed to different conditions and then fixed in 4% formaldehyde at room temperature for 5 min. They were then rinsed in PBS and permeabilized with 0.1% Triton X-100 in PBS prior to primary antibody exposition.
      Immunodetection of iNOS expression was performed using a rabbit anti-iNOS (1:100) (Sigma). After copious washes, the slides were then incubated for 1 h with a secondary goat anti-rabbit antibody conjugated to Alexa Fluor 594 (Invitrogen) diluted 1:500 in 0.005% (v/v) Triton X-100 in PBS.
      Immunodetection of 5,5-dimethylpyrroline-N-oxide (DMPO)-nitrone protein adducts on internalized parasites was performed using a rabbit anti-DMPO-nitrone serum that binds to the one-electron oxidation product of the initial DMPO nitroxyl protein spin adduct (
      • Detweiler C.D.
      • Deterding L.J.
      • Tomer K.B.
      • Chignell C.F.
      • Germolec D.
      • Mason R.P.
      ). Parasites were incubated with DMPO (100 mm) in dPBS for 30 min at 28 °C. After incubation, parasites were collected by centrifugation at 800 × g for 10 min at 25 °C and used in infection assays in Lab-Tek tissue culture chamber slides. Following macrophage infection (2 h) with preloaded trypomastigotes, cells fixed and permeabilized as described above were incubated for 30 min in a blocking solution containing 1% BSA in PBS, pH 7.4. Slides were then incubated for 1 h with the polyclonal anti-DMPO-nitrone (1:100). After three washes in 0.01% (v/v) Triton X-100 in PBS, secondary goat anti-rabbit antibody conjugated to Alexa Fluor 594 (Invitrogen) and diluted 1:500 in 0.005% (v/v) Triton X-100 in PBS was added for 1 h. Nuclei were visualized with DAPI (5 μg ml−1).
      For protein 3-nitrotyrosine immunodetection, fixed cells were blocked and incubated with rabbit anti-3-nitrotyrosine antisera produced in our laboratory (1:100) (
      • Brito C.
      • Naviliat M.
      • Tiscornia A.C.
      • Vuillier F.
      • Gualco G.
      • Dighiero G.
      • Radi R.
      • Cayota A.M.
      ) and, thereafter, with a goat anti-rabbit IgG antibody labeled with Alexa Fluor 488 (1:500). All slides were examined using an epifluorescence microscope.

      Transmission Electron Microscopy

      Infected macrophages were also prepared for examination by transmission electron microscopy. After the indicated incubation periods, the cells were fixed with a solution containing 4% (w/v) paraformaldehyde/0.2% (w/v) glutaraldehyde in PBS, pH 7.4, rinsed in the same buffer, and then post-fixed in 1% osmium tetroxide, dehydrated through graded ethanol solutions, and embedded in Araldite resin. Sections were made on an RMC MT-X Ultramicrotome with a DiATOME diamond knife. Ultrathin sections (40–60 nm) were stained with uranyl acetate and lead citrate and examined with a JEOL JEM-1010 electron microscope operating at 80 kV. The images were obtained with a Hamamatsu C-4742-95 digital camera and processed with the Photoimpact program.

      T. cruzi Cytotoxicity Assay

      T. cruzi metacyclic trypomastigotes were incubated for 24 h in 10 μCi ml−1 [5,6-3H]uridine at 28 °C. After incubation, the labeled parasites were collected by centrifugation at 800 × g and washed three times in dPBS, pH 7.4, to eliminate non-incorporated [5,6-3H]uridine. Macrophages were infected for 30 min at 37 °C with [5,6-3H]uridine-loaded metacyclic trypomastigotes (parasite:macrophage ratio of 5:1). Infection was synchronized by plate centrifugation (800 × g, 5 min), and following a 30-min incubation at 37 °C, cell supernatant containing non-internalized parasites (S1) was removed and conserved for quantification of the total count present in each condition. Macrophages were then washed three times with dPBS, pH 7.4, and after a 2-h incubation in DMEM at 37 °C, supernatants were collected (S2, killed parasites). Pelleted, infected macrophages were lysed in 200 μl of TET buffer (10 mm Tris/HCl, 1 mm EDTA, pH 8, with 0,5% Triton X-100). Cell pellets (P) and supernatants (S1 and S2) were each mixed with 3 ml of scintillation fluid, and radioactivity was measured in a liquid scintillation counter (Trilux 1450 Microbeta, Wallac Instruments). The results are expressed as a percentage of [5,6-3H]Uridine released during the 2-h infection period (S2) with respect to total [5,6-3H]uridine incorporation (p + S1 + S2) for each condition and represent the mean of three independent experiments.

      Determination of Parasite Load in Macrophages

      Macrophages were seeded in Lab-Tek tissue culture chamber slides and incubated for 5 h with or without iNOS inducers (IFN-γ plus LPS) before infection with metacyclic trypomastigotes (parasite:macrophage ratio of 5:1) for 1 h at 37 °C. Non-engulfed parasites were removed by washing twice in dPBS, and macrophages were further incubated for 24 h in DMEM at 37 °C. Infected macrophages were fixed in a 4% (v/v) formaldehyde solution in PBS for 10 min at room temperature, washed with PBS containing 100 mm glycine, and permeabilized for 5 min with 0.1% (v/v) Triton X-100 in PBS. The number of parasites per macrophage was determined by DAPI staining (5 μg ml−1). Preparations were analyzed using a microscope (Nikon Eclipse TE-200 inverted microscope) at ×1000 magnification, and digital photographs of infected macrophages were recorded. At least 2500 cells from five independent experiments were counted. Results are expressed as the number of amastigotes/100 macrophages under each condition and represent the mean of five independent experiments.

      Parasitemia and Histopathological Analysis

      BALB/c mice (n = 6) were infected with 1 × 106 metacyclic trypomastigotes by intraperitoneal injection with wild type parasites or T. cruzi overexpressing TcCPX. The blood (10 μl) of anesthetized mice was used to determine the parasite load and the number of trypomastigotes/100 fields of view every 10 days. After 60 days, tissue samples (heart and quadriceps muscle) of infected mice were recovered and fixed in 10% (v/v) fresh formaldehyde. Paraffin-embedded tissue sections (four nonconsecutive slides) from the heart and quadricep muscles of each mouse were stained with hematoxylin-eosin and examined under a light microscope (
      • Zago M.P.
      • Barrio A.B.
      • Cardozo R.M.
      • Duffy T.
      • Schijman A.G.
      • Basombrío M.A.
      ).

      Microscopy Analysis

      Imaging was performed using a Nikon Eclipse TE-200 inverted microscope coupled to a camera (SPOT RT camera, Diagnostic Instruments Inc.). Images were analyzed using ImageJ, a public domain Java image-processing program.

      Data Analysis

      Experiments were performed at least three times on independent days. All data are given as mean ± S.D. unless otherwise noted, and p < 0.05 was considered significant. Means were compared using the Student's t test. For comparison of more than two groups, an analysis of variance and a least-significant-difference post hoc test were performed.

      DISCUSSION

      During the course of the infection process, macrophages produce reactive oxygen and nitrogen species that are cytotoxic to several microbes including bacteria and parasites (
      • Hickman-Davis J.
      • Gibbs-Erwin J.
      • Lindsey J.R.
      • Matalon S.
      ,
      • Hurst J.K.
      • Lymar S.V.
      ,
      • Gookin J.L.
      • Allen J.
      • Chiang S.
      • Duckett L.
      • Armstrong M.U.
      ,
      • Vazquez-Torres A.
      • Jones-Carson J.
      • Balish E.
      ). The production of O2̇̄ depends on the activation of the NADPH oxidase complex, assembly of which is triggered by receptor-mediated phagocytosis. The precursor of reactive nitrogen species, NO, is produced after the cytokine-dependent induction of iNOS. This has led to the hypothesis (
      • Nathan C.
      • Shiloh M.U.
      ,
      • Alvarez M.N.
      • Piacenza L.
      • Irigoín F.
      • Peluffo G.
      • Radi R.
      ,
      • Evans T.J.
      • Buttery L.D.
      • Carpenter A.
      • Springall D.R.
      • Polak J.M.
      • Cohen J.
      ,
      • Fang F.C.
      ) that after microorganisms are ingested, O2̇̄ formed inside the phagocytic vacuole will react with NO diffusing from the cytosol to form microbicidal amounts of peroxynitrite. However, direct evidence for this phenomenon at the phagosomal level during microbial infection of macrophages has not been provided.
      The importance of oxidant production as the first line of defense of macrophages is underscored by the fact that some pathogens (
      • Mohapatra N.P.
      • Soni S.
      • Rajaram M.V.
      • Dang P.M.
      • Reilly T.J.
      • El-Benna J.
      • Clay C.D.
      • Schlesinger L.S.
      • Gunn J.S.
      ), mostly unicellular parasites, have evolved to evade the activation of these sets of enzymes in order to efficiently infect the host. For instance, Leishmania donovani promastigotes are internalized by macrophages without eliciting a respiratory burst, thereby avoiding O2̇̄ production (
      • Lodge R.
      • Diallo T.O.
      • Descoteaux A.
      ), and can also down-regulate macrophage NO production (
      • Damatta R.A.
      • Seabra S.H.
      • Deolindo P.
      • Arnholdt A.C.
      • Manhães L.
      • Goldenberg S.
      • de Souza W.
      ). Additionally, T. cruzi as well as other pathogenic trypanosomatids have an ample and well distributed set of antioxidant enzymes to cope with reactive oxygen and nitrogen species (
      • Piacenza L.
      • Peluffo G.
      • Alvarez M.N.
      • Kelly J.M.
      • Wilkinson S.R.
      • Radi R.
      ,
      • Wilkinson S.R.
      • Temperton N.J.
      • Mondragon A.
      • Kelly J.M.
      ,
      • Irigoín F.
      • Cibils L.
      • Comini M.A.
      • Wilkinson S.R.
      • Flohé L.
      • Radi R.
      ,
      • Krauth-Siegel R.L.
      • Comini M.A.
      ).
      Thus, to test our hypothesis, it was first essential to evaluate the influence of the infective forms of T. cruzi on the routes of O2̇̄ and NO formation and whether these radicals were formed simultaneously during an infection. In this context, the ability of T. cruzi trypomastigotes to trigger NADPH oxidase activity and promote the formation of significant levels of O2̇̄ was confirmed in infection experiments by three independent methods, namely NBT reduction, luminol oxidation, and cyanide-insensitive oxygen consumption (Fig. 1 and data under “Results”). Macrophage-dependent O2̇̄ production was responsive to the parasite load and was sustained over a 60–90-min period (Fig. 1B and oxygen consumption data under “Results”). Luminol, which equilibrates among intra- and extracellular compartments, and oxygen consumption data reflect overall cellular O2̇̄ production but do not discriminate the cellular sites (e.g. intraphagosomal versus extracellular) of O2̇̄ release. Thus, to assist in the site-specific detection of O2̇̄ in the phagosome of infected macrophages, NBT was used as a qualitative probe; the insoluble product from the O2̇̄-mediated reduction of NBT, formazan, was formed, deposited, and co-localized in the same cellular compartment where the internalized trypomastigotes were located (Fig. 1A). In regard to the NO pathway, non-infective epimastigotes inhibited NO production released by activated macrophages, as confirmed by evaluating both total NO2 accumulation and the time course of NO-dependent oxidation of DAF-2DA; this is consistent with iNOS mRNA levels and protein expression (Fig. 2) and in agreement with previous reports (
      • Stempin C.
      • Giordanengo L.
      • Gea S.
      • Cerbán F.
      ,
      • Stempin C.C.
      • Garrido V.V.
      • Dulgerian L.R.
      • Cerbán F.M.
      ). Importantly, this inhibitory effect did not operate for trypomastigotes that did not alter NO production from macrophages; interestingly, trypomastigotes caused some stimulation of the INF-γ-mediated iNOS induction (Fig. 2). The effect of the infective stage on iNOS expression could be attributed to TOLL-2 receptor (TLR2)- and CD-14-mediated recognition of trypomastigotes, which up-regulate iNOS expression (
      • Campos M.A.
      • Almeida I.C.
      • Takeuchi O.
      • Akira S.
      • Valente E.P.
      • Procópio D.O.
      • Travassos L.R.
      • Smith J.A.
      • Golenbock D.T.
      • Gazzinelli R.T.
      ,
      • Gazzinelli R.T.
      • Denkers E.Y.
      ,
      • Guiñazú N.
      • Pellegrini A.
      • Carrera-Silva E.A.
      • Aoki M.P.
      • Cabanillas A.M.
      • Gìronés N.
      • Fresno M.
      • Cano R.
      • Gea S.
      ,
      • Zhang S.
      • Kim C.C.
      • Batra S.
      • McKerrow J.H.
      • Loke P.
      ).
      Previous work by our group has provided evidence for the cytotoxic effect of macrophage-derived peroxynitrite to T. cruzi in model studies with the non-infective epimastigote stage (
      • Alvarez M.N.
      • Piacenza L.
      • Irigoín F.
      • Peluffo G.
      • Radi R.
      ,
      • Denicola A.
      • Rubbo H.
      • Rodríguez D.
      • Radi R.
      ,
      • Thomson L.
      • Denicola A.
      • Radi R.
      ,
      • Rubbo H.
      • Denicola A.
      • Radi R.
      ). In this study, we focused on the infective trypomastigote stage and the formation of peroxynitrite inside the phagosome of activated macrophages during parasite internalization. The capacity of peroxynitrite to cause oxidative damage to target cells is limited by its short half-life in the biological milieu and depends on the mean diffusion distance to its molecular targets (<10 ms, corresponding to <10 μm) (
      • Alvarez M.N.
      • Piacenza L.
      • Irigoín F.
      • Peluffo G.
      • Radi R.
      ). Electron microscope studies revealed that the distance between the parasite and the phagosomal membrane is quite small (determined to be ≤10–20 nm) (Fig. 5). Therefore, more than 90% of the peroxynitrite formed in the phagosomal lumen will reach parasite targets even in the presence of CO2 (
      • Alvarez M.N.
      • Piacenza L.
      • Irigoín F.
      • Peluffo G.
      • Radi R.
      ).
      Given its high concentration in biological fluids (1–2 mm), CO2 is a key reactant for peroxynitrite (k = 4.6 x 104 m −1 s−1, 37° C, pH 7.4), limiting its diffusion distance. The product of this reaction is the transient nitrosoperoxocarboxylate adduct that undergoes fast homolysis to CO3̇̄ and NO2 in ∼35% yields (
      • Ferrer-Sueta G.
      • Radi R.
      ).
      The previously reported lethal peroxynitrite dose for epimastigotes (
      • Alvarez M.N.
      • Piacenza L.
      • Irigoín F.
      • Peluffo G.
      • Radi R.
      ) is probably an underestimation when considering trypomastigotes because of the apparent increase in antioxidant enzymes observed during metacyclogenesis (
      • Piacenza L.
      • Peluffo G.
      • Alvarez M.N.
      • Kelly J.M.
      • Wilkinson S.R.
      • Radi R.
      ,
      • Piacenza L.
      • Zago M.P.
      • Peluffo G.
      • Alvarez M.N.
      • Basombrio M.A.
      • Radi R.
      ,
      • Atwood 3rd, J.A.
      • Weatherly D.B.
      • Minning T.A.
      • Bundy B.
      • Cavola C.
      • Opperdoes F.R.
      • Orlando R.
      • Tarleton R.L.
      ). The amount of intraphagosomal peroxynitrite formation was sufficient to kill trypomastigotes, as evidenced by protein nitroxidative modifications (Fig. 4), ultrastructural alterations (Fig. 5), and parasite killing (Table 1). Peroxynitrite stands as a significantly more potent cytotoxin against trypomastigotes than H2O2, as revealed by the lack of killing in unstimulated macrophages (Table 1). This is in good agreement with previous determinations with reagent peroxynitrite (
      • Alvarez M.N.
      • Piacenza L.
      • Irigoín F.
      • Peluffo G.
      • Radi R.
      ) and H2O2 (
      • Tanaka Y.
      • Tanowitz H.
      • Bloom B.R.
      ), respectively.
      Protein oxidative damage in phagocytosed trypomastigotes was detected by immunostaining with anti-DMPO-nitrone and anti 3-nitrotyrosine antibodies; this revealed modified protein in close proximity to T. cruzi nuclei and kinetoplasts, which further supports the phagosomal generation of peroxynitrite (Fig. 4). This result is in contrast to that reported during the infection of macrophages by Salmonella typhimurium. In this case, the bacteria did not co-localize with protein 3-nitrotyrosine because it interfered with NADPH oxidase activation, thereby preventing O2̇̄ and peroxynitrite formation in the vesicle (
      • Vazquez-Torres A.
      • Jones-Carson J.
      • Mastroeni P.
      • Ischiropoulos H.
      • Fang F.C.
      ,
      • Chakravortty D.
      • Hansen-Wester I.
      • Hensel M.
      ). Alternative systems to account for T. cruzi protein tyrosine nitration, such as MPO-H2O2-NO2 (
      • Radi R.
      ,
      • Pfeiffer S.
      • Lass A.
      • Schmidt K.
      • Mayer B.
      ,
      • Eiserich J.P.
      • Hristova M.
      • Cross C.E.
      • Jones A.D.
      • Freeman B.A.
      • Halliwell B.
      • van der Vliet A.
      ,
      • Cape J.L.
      • Hurst J.K.
      ,
      • Gomez-Mejiba S.E.
      • Zhai Z.
      • Gimenez M.S.
      • Ashby M.T.
      • Chilakapati J.
      • Kitchin K.
      • Mason R.P.
      • Ramirez D.C.
      ), were not operative due to the lack of MPO in J774A-1 macrophages (supplemental Fig. 1). Peroxidase activity in J774A-1 macrophages may arise from cyclooxygenase-2 induction, but this may be relevant only at 15–20 h post-activation (
      • Palazzolo-Ballance A.M.
      • Suquet C.
      • Hurst J.K.
      ), a time frame beyond the experiments reported herein and in full agreement with the lack of oxidative effects on T. cruzi upon macrophage incubation with NO2 (supplemental Fig. 2).
      To further evaluate the role of peroxynitrite toxicity and its relationship to the control of macrophage infection, we used chemically transformed trypomastigotes overexpressing TcCPX as a molecular tool; previous work had shown that this enzyme can efficiently detoxify peroxynitrite in T. cruzi (
      • Piacenza L.
      • Peluffo G.
      • Alvarez M.N.
      • Kelly J.M.
      • Wilkinson S.R.
      • Radi R.
      ). Although some data on peroxiredoxin activity toward peroxynitrite in biochemical and cellular systems have been provided (
      • Piacenza L.
      • Peluffo G.
      • Alvarez M.N.
      • Kelly J.M.
      • Wilkinson S.R.
      • Radi R.
      ,
      • Trujillo M.
      • Budde H.
      • Piñeyro M.D.
      • Stehr M.
      • Robello C.
      • Flohé L.
      • Radi R.
      ,
      • Bryk R.
      • Griffin P.
      • Nathan C.
      ,
      • Barr S.D.
      • Gedamu L.
      ,
      • Master S.S.
      • Springer B.
      • Sander P.
      • Boettger E.C.
      • Deretic V.
      • Timmins G.S.
      ), direct and unambiguous confirmation of its capacity to prevent peroxynitrite-mediated effects during microbial infection to mammalian cells was lacking. We found that RH 123 formation on phagocytosed DHR-loaded wild type trypomastigotes depended on peroxynitrite formation and that the observed fluorescence was confined to the phagosome (Fig. 3). Parasites with elevated levels of TcCPX showed less DHR oxidation, in agreement with the increased ability of cells to decompose peroxynitrite (Fig. 6A). Importantly, activated macrophages capable of simultaneously producing O2̇̄ and NO during internalization of wild type trypomastigotes limited the progression of the infection, causing a decrease of 60% in parasite load when compared with macrophages producing only O2̇̄ or NO, or even both, but with a temporal mismatch (Table 1). Ultrastructural analysis of phagocytosed parasites by peroxynitrite producing activated macrophages showed marked damage of cellular organelles and disruption of cell membrane (Fig. 5). TcCPX-overexpressing trypomastigotes resisted cytotoxicity as evaluated by a 3[H]uridine release assay (Fig. 6B), which is compatible with an efficient neutralization of peroxynitrite, resulting in higher infection yields at 24 h post-infection (Table 1). In line with this finding, experimental infections of mice with trypomastigotes having higher levels of TcCPX resulted in higher parasitemias and tissue inflammation compared with the infection with wild type parasites (Fig. 7). These observations recapitulated well the previous findings of our group, showing a positive association between the levels of peroxiredoxins in several parasite strains and their degree of virulence (
      • Piacenza L.
      • Zago M.P.
      • Peluffo G.
      • Alvarez M.N.
      • Basombrio M.A.
      • Radi R.
      ) The role of NO in the control of T. cruzi infection in vivo (
      • Gutierrez F.R.
      • Mineo T.W.
      • Pavanelli W.R.
      • Guedes P.M.
      • Silva J.S.
      ) and the fact that parasite peroxiredoxins do not decompose NO, but rather NO-derived peroxynitrite, fully support the notion that peroxynitrite acts as a cytotoxin in vivo and the existence of an interplay between peroxynitrite and peroxiredoxins in influencing the outcome of infection (
      • Piacenza L.
      • Alvarez M.N.
      • Peluffo G.
      • Radi R.
      ).
      In summary, the present work demonstrates that peroxynitrite is generated within the phagosome of activated macrophages from the reaction of O2̇̄, arising from the activation of NADPH oxidase secondary to internalization of the parasite, with NO diffusing from the cytosol. Peroxynitrite was formed for about 60–90 min and in sufficient amounts to diffuse into the parasite and to cause substantial cytotoxicity, to a much larger extent than that of its precursor radicals and H2O2. The effects of peroxynitrite were neutralized by the overexpression of TcCPX in the parasites, which facilitated the development of the infection. The dynamic balance between intraphagosomal oxidant production and the activity of key antioxidant enzymes in the parasite at the onset of the infection critically determined parasite infectivity, providing additional insights for the differences observed on natural T. cruzi strains of diverse virulence and pathogenicity in the human disease (
      • Piacenza L.
      • Zago M.P.
      • Peluffo G.
      • Alvarez M.N.
      • Basombrio M.A.
      • Radi R.
      ).

      Acknowledgments

      We thank Dr. Shane R. Wilkinson (School of Biological and Chemical Sciences, Queen Mary University of London, UK) for critical reading of the manuscript and generously providing T. cruzi epimastigotes overexpressing cytosolic TcCPX and Dr. Ronald Mason (NIEHS, National Institutes of Health, Research Triangle Park, NC) for the anti-DMPO-nitrone serum. We are grateful to Dr. Gabriela Casanova (Unidad de Microscopía Electrónica, Facultad de Ciencias, Universidad de la República, Uruguay) for help with transmission electron microscopy studies, Dr. Paola Zago and Dr. Miguel Basombrio (Instituto de Patología Experimental, Facultad de Ciencias de la Salud, UNSa-CONICET, Salta, Argentina) for assistance in the preparation of tissue sections, and Cristina Oviedo (Universidad de la República, Uruguay) for assistance with experimental infections.

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