Carolina Prolo

Peroxynitrite, a potent macrophage‐derived oxidizing cytotoxin to combat invading pathogens

Macrophages are among the first cellular actors facing the invasion of microorganisms. These cells are able to internalize pathogens and destroy them by means of toxic mediators, many of which are produced enzymatically and have strong oxidizing capacity. Indeed, macrophages count on the NADPH oxidase complex activity, which is triggered during pathogen invasion and leads to the production of superoxide radical inside the phagosome. At the same time, the induction of nitric oxide synthase results in the production of nitric oxide in the cytosol which is able to readily diffuse to the phagocytic vacuole. Superoxide radical and nitric oxide react at diffusion controlled rates with each other inside the phagosome to yield peroxynitrite, a powerful oxidant capable to kill micro‐organisms. Peroxynitrite toxicity resides on oxidations and nitrations of biomolecules in the target cell. The central role of peroxynitrite as a key effector molecule in the control of infections has been proven in a wide number of models. However, some microorganisms and virulent strains adapt to survive inside the potentially hostile oxidizing microenvironment of the phagosome by either impeding peroxynitrite formation or rapidly detoxifying it once formed. In this context, the outcome of the infection process is a result of the interplay between the macrophage‐derived oxidizing cytotoxins such as peroxynitrite and the antioxidant defense machinery of the invading pathogens.

Nitroarachidonic acid prevents NADPH oxidase assembly and superoxide radical production in activated macrophages.

Nitration of arachidonic acid (AA) to nitroarachidonic acid (AANO2) leads to anti-inflammatory intracellular activities during macrophage activation. However, less is known about the capacity of AANO2 to regulate the production of reactive oxygen species under proinflammatory conditions. One of the immediate responses upon macrophage activation involves the production of superoxide radical (O2(•-)) due to the NADPH-dependent univalent reduction of oxygen to O2(•-) by the phagocytic NADPH oxidase isoform (NOX2), the activity of NOX2 being the main source of O2(•-) in monocytes/macrophages. Because the NOX2 and AA pathways are connected, we propose that AANO2 can modulate macrophage activation by inhibiting O2(•-) formation by NOX2. When macrophages were activated in the presence of AANO2, a significant inhibition of NOX2 activity was observed as evaluated by cytochrome c reduction, luminol chemiluminescence, Amplex red fluorescence, and flow cytometry; this process also occurs under physiological mimic conditions within the phagosomes. AANO2 decreased O2(•-) production in a dose- (IC50=4.1±1.8 μM AANO2) and time-dependent manner. The observed inhibition was not due to a decreased phosphorylation of the cytosolic subunits (e.g., p40(phox) and p47(phox)), as analyzed by immunoprecipitation and Western blot. However, a reduction in the migration to the membrane of p47(phox) was obtained, suggesting that the protective actions involve the prevention of the correct assembly of the active enzyme in the membrane. Finally, the observed in vitro effects were confirmed in an in vivo inflammatory model, in which subcutaneous injection of AANO2 was able to decrease NOX2 activity in macrophages from thioglycolate-treated mice.

Sensitive Detection and Estimation of Cell-Derived Peroxynitrite Fluxes Using Fluorescein-Boronate

The specific and sensitive detection of peroxynitrite (ONOO-/ONOOH) in biological systems is a great challenge due to its high reactivity towards several biomolecules. Herein, we validated the advantages of using fluorescein-boronate (Fl-B) as a highly sensitive fluorescent probe for the direct detection of peroxynitrite under biologically-relevant conditions in two different cell models. The synthesis of Fl-B was achieved by a very simply two-step conversion synthetic route with high purity (>99%) and overall yield (∼42%). Reactivity analysis of Fl-B with relevant biological oxidants including hydrogen peroxide (H2O2), hypochlorous acid (HOCl) and peroxynitrite were performed. The rate constant for the reaction of peroxynitrite with Fl-B was 1.7×106M-1s-1, a million times faster than the rate constant measured for H2O2 (k=1.7M-1s-1) and 2,700 faster than HOCl (6.2×102M-1s-1) at 37°C and pH 7.4. The reaction of Fl-B with peroxynitrite was significant even in the presence of physiological concentrations of CO2, a well-known peroxynitrite reactant. Experimental and simulated kinetic analyses confirm that the main oxidation process of Fl-B takes place with peroxynitrite itself via a direct bimolecular reaction and not with peroxynitrite-derived radicals. Fl-B was successfully applied for the detection of endogenously-generated peroxynitrite by endothelial cells and in macrophage-phagocyted parasites. Moreover, the generated data allowed estimating the actual intracellular flux of peroxynitrite. For instance, ionomycin-stimulated endothelial cells generated peroxynitrite at a rate of ∼ 0.1μMs-1, while immunostimulated macrophages do so in the order of ∼1μMs-1 inside T. cruzi-infected phagosomes. Fl-B revealed not to be toxic in concentrations up to 1mM for 24h. Cellular peroxynitrite detection was achieved by conventional laboratory fluorescence-based methods including flow cytometry and epi-fluorescence microscopy. Fl-B was shown to be more sensitive than the coumarin boronate due to a higher molar absorption coefficient and quantum yield. Overall, our results show that Fl-B is a kinetically selective and highly sensitive probe for the direct detection of cell-derived peroxynitrite.

Nitric oxide diffusion to red blood cells limits extracellular, but not intraphagosomal, peroxynitrite formation by macrophages

Macrophage-derived nitric oxide ((•)NO) participates in cytotoxic mechanisms against diverse microorganisms and tumor cells. These effects can be mediated by (•)NO itself or (•)NO-derived species such as peroxynitrite formed by its diffusion-controlled reaction with NADPH oxidase-derived superoxide radical anion (O(2)(•-)). In vivo, the facile extracellular diffusion of (•)NO as well as different competing consumption routes limit its bioavailability for the reaction with O(2)(•-) and, hence, peroxynitrite formation. In this work, we evaluated the extent by which (•)NO diffusion to red blood cells (RBC) can compete with activated macrophages-derived O(2)(•-) and affect peroxynitrite formation yields. Macrophage-dependent peroxynitrite production was determined by boron-based probes that react directly with peroxynitrite, namely, coumarin-7-boronic acid (CBA) and fluorescein-boronate (Fl-B). The influence of (•)NO diffusion to RBC on peroxynitrite formation was experimentally analyzed in co-incubations of (•)NO and O(2)(•-)-forming macrophages with erythrocytes. Additionally, we evaluated the permeation of (•)NO to RBC by measuring the intracellular oxidation of oxyhemoglobin to methemoglobin. Our results indicate that diluted RBC suspensions dose-dependently inhibit peroxynitrite formation, outcompeting the O(2)(•-) reaction. Computer-assisted kinetic studies evaluating peroxynitrite formation by its precursor radicals in the presence of RBC are in accordance with experimental results. Moreover, the presence of erythrocytes in the proximity of (•)NO and O(2)(•-)-forming macrophages prevented intracellular Fl-B oxidation pre-loaded in L1210 cells co-cultured with activated macrophages. On the other hand, Fl-B-coated latex beads incorporated in the macrophage phagocytic vacuole indicated that intraphagosomal probe oxidation by peroxynitrite was not affected by nearby RBC. Our data support that in the proximity of a blood vessel, (•)NO consumption by RBC will limit the extracellular formation (and subsequent cytotoxic effects) of peroxynitrite by activated macrophages, while the intraphagosomal yield of peroxynitrite will remain unaffected.

Fluorescence and chemiluminescence approaches for peroxynitrite detection

ABSTRACT In the last two decades, there has been a significant advance in understanding the biochemistry of peroxynitrite, an endogenously‐produced oxidant and nucleophile. Its relevance as a mediator in several pathologic states and the aging process together with its transient character and low steady‐state concentration, motivated the development of a variety of techniques for its unambiguous detection and estimation. Among these, fluorescence and chemiluminescence approaches have represented important tools with enhanced sensitivity but usual limited specificity. In this review, we analyze selected examples of molecular probes that permit the detection of peroxynitrite by fluorescence and chemiluminescence, disclosing their mechanism of reaction with either peroxynitrite or peroxynitrite‐derived radicals. Indeed, probes have been divided into 1) redox probes that yield products by a free radical mechanism, and 2) electrophilic probes that evolve to products secondary to the nucleophilic attack by peroxynitrite. Overall, boronate‐based compounds are emerging as preferred probes for the sensitive and specific detection and quantitation. Moreover, novel strategies involving genetically‐modified fluorescent proteins with the incorporation of unnatural amino acids have been recently described as peroxynitrite sensors. This review analyzes the most commonly used fluorescence and chemiluminescence approaches for peroxynitrite detection and provides some guidelines for appropriate experimental design and data interpretation, including how to estimate peroxynitrite formation rates in cells. Graphical abstract Figure. No Caption available. HighlightsPeroxynitrite can be detected by fluorescent and chemiluminescent probes.Probes can react with peroxynitrite by redox‐ or nucleophilic‐based mechanisms.Current approaches allow peroxynitrite visualization in cells and tissues.Boronate‐based chemical probes are highly selective for peroxynitrite detection.Genetically‐encoded probes with reactive unnatural amino acids are being developed.

Peroxidase, holdase and signaling functions of Trypanosoma cruzi mitochondrial peroxiredoxin

The etiological agent of Chagas disease, Trypanosoma cruzi contains 2-Cys peroxiredoxins (Prx) localized in mitochondria (TcMPX) and cytosol (TcCPX) as part of its antioxidant armamentarium. The participation of these Prx in the defense towards the host and parasite-derived oxidative stress led us to postulate them as virulence factors. In addition to its peroxidase activity, 2-Cys Prx have alternative functions. In this work, we studied the peroxidase, holdase and signaling functions of TcMPX in the context of parasite-macrophage and drug interactions (nifurtimox, NFX). Parasites overexpressing TcMPX were more infective compared to wild type (wt) under macrophage-derived O2•- and/or ONOO- generation indicating the protective role of TcMPX during infection. Additionally, TcMPX overexpressers also contain higher holdase activity and were more resistant to NFX-treatment. Importantly, wt parasites treated with NFX increase TcMPX protein content suggesting the involvement of the holdase activity in the stress response towards NFX. Finally, under stress conditions, TcMPX protein-disulfide adducts were detected indicating additional redox protein partners which may participate in the biological actions of TcMPX.

Peroxynitrite Formation and Detection in Living Cells

Abstract Different cell types produce peroxynitrite under conditions of simultaneous generation of its precursor radicals, superoxide and nitric oxide. The detection of cell-derived peroxynitrite is technically challenging due to its short biological half-life and low steady-state concentration and the shortage of specific methods. However, appropriate use of molecular footprints and probes together with a wise application of pharmacological and genetic approaches, allow for its unambiguous detection. Among the molecular footprints left by peroxynitrite, the measurement and identification of tyrosine-nitrated proteins is of prime relevance. Regarding molecular probes, recent advances on the characterization of the reactivity of boronate-based molecules with peroxynitrite have opened new and more specific ways for cellular detection. A critical analysis of the chemical basis and usefulness of the existing methods utilized for the cellular detection of peroxynitrite will be performed. Also, the role of intracellular modulators of peroxynitrite reactivity and levels (e.g., CO2, uric acid, peroxiredoxins) and how they can influence the detected levels will be assessed. The accurate cellular detection of peroxynitrite in different cellular and extracellular compartments and the estimation of its formation rates, represent fundamental steps to understand how nitric oxide-derived oxidants affect biological processes, including mitochondrial dysfunction and cell death.