Marcus F. Oliveira

Characterization of Heme as Activator of Toll-like Receptor 4

Heme is an ancient and ubiquitous molecule present in organisms of all kingdoms, composed of an atom of iron linked to four ligand groups of porphyrin. A high amount of free heme, a potential amplifier of the inflammatory response, is a characteristic feature of diseases with increased hemolysis or extensive cell damage. Here we demonstrate that heme, but not its analogs/precursors, induced tumor necrosis factor-α (TNF-α) secretion by macrophages dependently on MyD88, TLR4, and CD14. The activation of TLR4 by heme is exquisitely strict, requiring its coordinated iron and the vinyl groups of the porphyrin ring. Signaling of heme through TLR4 depended on an interaction distinct from the one established between TLR4 and lipopolysaccharide (LPS) since anti-TLR4/MD2 antibody or a lipid A antagonist inhibited LPS-induced TNF-α secretion but not heme activity. Conversely, protoporphyrin IX antagonized heme without affecting LPS-induced activation. Moreover, heme induced TNF-α and keratinocyte chemokine but was ineffective to induce interleukin-6, interleukin-12, and interferon-inducible protein-10 secretion or co-stimulatory molecule expression. These findings support the concept that the broad ligand specificity of TLR4 and the different activation profiles might in part reside in its ability to recognize different ligands in different binding sites. Finally, heme induced oxidative burst, neutrophil recruitment, and heme oxygenase-1 expression independently of TLR4. Thus, our results presented here reveal a previous unrecognized role of heme as an extracellular signaling molecule that affects the innate immune response through a receptor-mediated mechanism.

Mitochondrial Creatine Kinase Activity Prevents Reactive Oxygen Species Generation ANTIOXIDANT ROLE OF MITOCHONDRIAL KINASE-DEPENDENT ADP RE-CYCLING ACTIVITY

As recently demonstrated by our group (da-Silva, W. S., Gómez-Puyou, A., Gómez-Puyou, M. T., Moreno-Sanchez, R., De Felice, F. G., de Meis, L., Oliveira, M. F., and Galina, A. (2004) J. Biol. Chem. 279, 39846–39855) mitochondrial hexokinase activity (mt-HK) plays a preventive antioxidant role because of steady-state ADP re-cycling through the inner mitochondrial membrane in rat brain. In the present work we show that ADP re-cycling accomplished by the mitochondrial creatine kinase (mt-CK) regulates reactive oxygen species (ROS) generation, particularly in high glucose concentrations. Activation of mt-CK by creatine (Cr) and ATP or ADP, induced a state 3-like respiration in isolated brain mitochondria and prevention of H2O2 production obeyed the steady-state kinetics of the enzyme to phosphorylate Cr. The extension of the preventive antioxidant role of mt-CK depended on the phosphocreatine (PCr)/Cr ratio. Rat liver mitochondria, which lack mt-CK activity, only reduced state 4-induced H2O2 generation when 1 order of magnitude more exogenous CK activity was added to the medium. Simulation of hyperglycemic conditions, by the inclusion of glucose 6-phosphate in mitochondria performing 2-deoxyglucose phosphorylation via mt-HK, induced H2O2 production in a Cr-sensitive manner. Simulation of hyperglycemia in embryonic rat brain cortical neurons increased both ΔΨm and ROS production and both parameters were decreased by the previous inclusion of Cr. Taken together, the results presented here indicate that mitochondrial kinase activity performed a key role as a preventive antioxidant against oxidative stress, reducing mitochondrial ROS generation through an ADP-recycling mechanism.

Platelets mediate increased endothelium permeability in dengue through NLRP3-inflammasome activation

Dengue is the most frequent hemorrhagic viral disease and re-emergent infection in the world. Although thrombocytopenia is characteristically observed in mild and severe forms of dengue, the role of platelet activation in dengue pathogenesis has not been fully elucidated. We hypothesize that platelets have major roles in inflammatory amplification and increased vascular permeability during severe forms of dengue. Here we investigate interleukin (IL)-1β synthesis, processing, and secretion in platelets during dengue virus (DV) infection and potential contribution of these events to endothelial permeability during infection. We observed increased expression of IL-1β in platelets and platelet-derived microparticles from patients with dengue or after platelet exposure to DV in vitro. We demonstrated that DV infection leads to assembly of nucleotide-binding domain leucine rich repeat containing protein (NLRP3) inflammasomes, activation of caspase-1, and caspase-1-dependent IL-1β secretion. Our findings also indicate that platelet-derived IL-1β is chiefly released in microparticles through mechanisms dependent on mitochondrial reactive oxygen species-triggered NLRP3 inflammasomes. Inflammasome activation and platelet shedding of IL-1β-rich microparticles correlated with signs of increased vascular permeability. Moreover, microparticles from DV-stimulated platelets induced enhanced permeability in vitro in an IL-1-dependent manner. Our findings provide new evidence that platelets contribute to increased vascular permeability in DV infection by inflammasome-dependent release of IL-1β.

Haemozoin in Schistosoma mansoni.

a Departamento de Bioquimica Medica, Uni6ersidade Federal do Rio de Janeiro, Cidade Uni6ersitaria, 21941-590, Rio de Janeiro, RJ, Brazil b Di6isao de Quimica, Setor de Quimica Orgânica, CENPES, Petrobras, Cidade Uni6ersitaria, 21949-900, Rio de Janeiro, RJ, Brazil c Centro de Biociencias e Biotecnologia, Uni6ersidade Estadual do Norte Fluminense, Campos dos Goytacazes, RJ, 28015-620, Brazil d Instituto de Biofisica Carlos Chagas Filho, Uni6ersidade Federal do Rio de Janeiro, Cidade Uni6ersitaria, 21941-590, Rio de Janeiro, RJ, Brazil

Haem detoxification by an insect

Haem is involved in many biological reactions, including oxygen transport, respiration and photosynthesis. In the free state, however, haem can generate reactive oxygen species that can damage biological molecules. It can also disrupt the phospholipid bilayer of cell membranes. In Plasmodium parasites, which are the aetiological agents of malaria disease, up to 80% of host-cell haemoglobin is digested, leaving the free haem group to be detoxified in the parasites food vacuole by polymerizing it into a harmless dark-brown crystalline structure called malaria pigment or haemozoin. Haem detoxification is also a challenge for blood-sucking insects, which digest several times their own weight of vertebrate blood during a blood meal. Here we show that haem polymerization into haemozoin is not exclusive to Plasmodium: it also occurs in the midgut of the blood-sucking insect Rhodnius prolixus(Hemiptera), an important vector of Trypanosoma cruzi, the causative agent of Chagas’ disease.

Sepsis induces brain mitochondrial dysfunction.

Objective:Mitochondrial dysfunctions have been associated with the pathogenesis of sepsis. A systematic survey of mitochondrial function in brain tissues during sepsis is lacking. In the present work, we investigate brain mitochondrial function in a septic mouse model. Design:Prospective animal study. Setting:University research laboratory. Subjects:Male Swiss mice, aged 6–8 wks. Interventions:Mice were subjected to cecal ligation and perforation (sepsis group) with saline resuscitation or to sham operation (control group). Measurements and Main Results:Oxygen consumption was measured polarographically in an oximeter. Brain homogenates from septic animals presented higher oxygen consumption in the absence of adenosine 5′-diphosphate (state 4) compared with control animals. The increase in state 4 respiration in animals in the cecal ligation and perforation group resulted in a drastic decrease in both respiratory control and adenosine 5′-diphosphate/oxygen ratios, indicating a reduction in the oxidative phosphorylation efficiency. Septic animals presented a significant increase in the recovery time of mitochondrial membrane potential on adenosine 5′-diphosphate addition compared with control animals, suggesting a proton leak through the inner mitochondrial membrane. The septic group presented a general reduction in the content of cytochromes. Moreover, the activity of cytochrome c oxidase was specifically and significantly decreased in the brain during sepsis. Hydrogen peroxide generation by brain mitochondria from septic mice did not respond to substrates of electron transport chain or to adenosine 5′-diphosphate, showing that mitochondrial function may be compromised in a critical level in the brain during sepsis. Conclusions:The mitochondrial dysfunctions demonstrated here indicate that uncoupling of oxidative phosphorylation takes place in the brain of septic mice, compromising tissue bioenergetic efficiency.

Structural and morphological characterization of hemozoin produced by Schistosoma mansoni and Rhodnius prolixus

Hemozoin (Hz) is a heme crystal produced upon the digestion of hemoglobin (Hb) by blood‐feeding organisms as a main mechanism of heme disposal. The structure of Hz consists of heme dimers bound by reciprocal iron–carboxylate interactions and stabilized by hydrogen bonds. We have recently described heme crystals in the blood fluke, Schistosoma mansoni, and in the kissing bug, Rhodnius prolixus. Here, we characterized the structures and morphologies of the heme crystals from those two organisms and compared them to synthetic β‐hematin (βH). Synchrotron radiation X‐ray powder diffraction showed that all heme crystals share the same unit cell and structure. The heme crystals isolated from S. mansoni and R. prolixus consisted of very regular units assembled in multicrystalline spherical structures exhibiting remarkably distinct surface morphologies compared to βH. In both organisms, Hz formation occurs inside lipid droplet‐like particles or in close association to phospholipid membranes. These results show, for the first time, the structural and morphological characterization of natural Hz samples obtained from these two blood‐feeding organisms. Moreover, Hz formation occurring in close association to a hydrophobic environment seems to be a common trend for these organisms and may be crucial to produce very regular shaped phases, allowing the formation of multicrystalline assemblies in the guts of S. mansoni and R. prolixus.

Dengue induces platelet activation, mitochondrial dysfunction and cell death through mechanisms that involve DC-SIGN and caspases

Worldwide, dengue is the most prevalent human arbovirus disease. Dengue infection may cause a range of clinical manifestations from self‐limiting febrile illness through to a life‐threatening syndrome accompanied by both bleeding and shock. Thrombocytopenia is frequently observed in mild and severe disease; however, the mechanisms involved in DENV‐induced platelet activation and thrombocytopenia are incompletely understood.

Cognitive Dysfunction Is Sustained after Rescue Therapy in Experimental Cerebral Malaria, and Is Reduced by Additive Antioxidant Therapy

Neurological impairments are frequently detected in children surviving cerebral malaria (CM), the most severe neurological complication of infection with Plasmodium falciparum. The pathophysiology and therapy of long lasting cognitive deficits in malaria patients after treatment of the parasitic disease is a critical area of investigation. In the present study we used several models of experimental malaria with differential features to investigate persistent cognitive damage after rescue treatment. Infection of C57BL/6 and Swiss (SW) mice with Plasmodium berghei ANKA (PbA) or a lethal strain of Plasmodium yoelii XL (PyXL), respectively, resulted in documented CM and sustained persistent cognitive damage detected by a battery of behavioral tests after cure of the acute parasitic disease with chloroquine therapy. Strikingly, cognitive impairment was still present 30 days after the initial infection. In contrast, BALB/c mice infected with PbA, C57BL6 infected with Plasmodium chabaudi chabaudi and SW infected with non lethal Plasmodium yoelii NXL (PyNXL) did not develop signs of CM, were cured of the acute parasitic infection by chloroquine, and showed no persistent cognitive impairment. Reactive oxygen species have been reported to mediate neurological injury in CM. Increased production of malondialdehyde (MDA) and conjugated dienes was detected in the brains of PbA-infected C57BL/6 mice with CM, indicating high oxidative stress. Treatment of PbA-infected C57BL/6 mice with additive antioxidants together with chloroquine at the first signs of CM prevented the development of persistent cognitive damage. These studies provide new insights into the natural history of cognitive dysfunction after rescue therapy for CM that may have clinical relevance, and may also be relevant to cerebral sequelae of sepsis and other disorders.

The effects on Trypanosoma cruzi of novel synthetic naphthoquinones are mediated by mitochondrial dysfunction.

Despite ongoing efforts, the current treatment for Chagas disease is still unsatisfactory, mainly because of the severe side effects and variable efficacy of the available nitroheterocycles. Our group has been assaying natural quinones isolated from Brazilian flora, and their derivatives, as alternative chemotherapeutic agents against Trypanosoma cruzi. From C-allyl lawsone three naphthofuranquinones were synthesized, which were active against trypomastigotes and epimastigotes. Here, we further investigated the activity and the mechanisms of action of these quinones. They exhibited powerful effects on intracellular amastigotes, presenting low toxicity to the host cells. Ultrastructural analyses of treated epimastigotes and trypomastigotes indicated a potent effect of the three naphthofuranquinones on the parasite mitochondrion, which appeared drastically swollen and with a washed-out matrix profile. Fluorescence-activated cell sorting analysis of rhodamine 123-stained T. cruzi showed that the three naphthofuranquinones caused a potent dose-dependent collapse of the mitochondrial membrane potential, especially in the epimastigote form. Naphthofuranquinones also decreased specifically mitochondrial complex I-III activity in both epimastigotes and trypomastigotes, parallel to a reduction in succinate-induced oxygen consumption. Mitochondrial hydrogen peroxide formation was also increased in epimastigotes after treatment with the naphthofuranquinones. Our results indicate that the trypanocidal action of the naphthofuranquinones is associated with mitochondrial dysfunction, leading to increased reactive oxygen species generation and parasite death.