Oleksii A. Skorokhod

Hemozoin (Malarial Pigment) Inhibits Differentiation and Maturation of Human Monocyte-Derived Dendritic Cells: A Peroxisome Proliferator-Activated Receptor-γ-Mediated Effect

Acute and chronic Plasmodium falciparum malaria are accompanied by severe immunodepression possibly related to subversion of dendritic cells (DC) functionality. Phagocytosed hemozoin (malarial pigment) was shown to inhibit monocyte functions related to immunity. Hemozoin-loaded monocytes, frequently found in circulation and adherent to endothelia in malaria, may interfere with DC development and play a role in immunodepression. Hemozoin-loaded and unloaded human monocytes were differentiated in vitro to immature DC (iDC) by treatment with GM-CSF and IL-4, and to mature DC (mDC) by LPS challenge. In a second setting, hemozoin was fed to iDC further cultured to give mDC. In both settings, cells ingested large amounts of hemozoin undegraded during DC maturation. Hemozoin-fed monocytes did not apoptose but their differentiation and maturation to DC was severely impaired as shown by blunted expression of MHC class II and costimulatory molecules CD83, CD80, CD54, CD40, CD1a, and lower levels of CD83-specific mRNA in hemozoin-loaded iDC and mDC compared with unfed or latex-loaded DC. Further studies indicated activation of peroxisome proliferator-activated receptor-γ (PPAR-γ) in hemozoin-loaded iDC and mDC, associated with increased expression of PPAR-γ mRNA, without apparent involvement of NF-κB. Moreover, expression of PPAR-γ was induced and up-regulation of CD83 was inhibited by supplementing iDC and mDC with plausible concentrations of 15(S)-hydroxyeicosatetraenoic acid, a PPAR-γ ligand abundantly produced by hemozoin via heme-catalyzed lipoperoxidation.

Host fibrinogen stably bound to hemozoin rapidly activates monocytes via TLR-4 and CD11b/CD18-integrin: a new paradigm of hemozoin action

Natural hemozoin (nHZ), prepared after schizogony, consists of crystalline ferriprotoporphyrin-IX dimers from undigested heme bound to host and parasite proteins and lipids. Phagocytosed nHZ alters important functions of host phagocytes. Most alterations are long-term effects. We show that host fibrinogen (FG) was constantly present (at ~ 1 FG per 25 000 HZ-heme molecules) and stably bound to nHZ from plasma-cultured parasites. FG was responsible for the rapid 100-fold stimulation of reactive oxygen species production and 50-fold increase of TNF and monocyte chemotactic protein 1 by human monocytes. Those effects, starting within minutes after nHZ cell contact, were because of interaction of FG with FG-receptors TLR4 and integrin CD11b/CD18. Receptor blockage by specific mAbs or removal of FG from nHZ abrogated the effects. nHZ-opsonizing IgGs contribute to the stimulatory response but are not essential for FG effects. Immediate increase in reactive oxygen species and TNF may switch on previously described long-term effects of nHZ, largely because of HZ-generated lipo-peroxidation products 15(S,R)-hydroxy-6,8,11,13-eicosatetraenoic acid and 4-hydroxynonenal. The FG/HZ effects mediated by TLR4/integrins represent a novel paradigm of nHZ activity and allow expansion of nHZ effects to nonphagocytic cells, such as endothelia and airway epithelia, and lead to a better understanding of organ pathology in malaria.

Inhibition of erythropoiesis in malaria anemia: role of hemozoin and hemozoin-generated 4-hydroxynonenal.

Severe malaria anemia is characterized by inhibited/altered erythropoiesis and presence of hemozoin-(HZ)-laden bone-marrow macrophages. HZ mediates peroxidation of unsaturated fatty acids and production of bioactive aldehydes such as 4-hydroxynonenal (HNE). HZ-laden human monocytes inhibited growth of cocultivated human erythroid cells and produced HNE that diffused to adjacent cells generating HNE-protein adducts. Cocultivation with HZ or treatment with low micromolar HNE inhibited growth of erythroid cells interfering with cell cycle without apoptosis. After HZ/HNE treatment, 2 critical proteins in cell-cycle regulation, p53 and p21, were increased and the retinoblastoma protein, central regulator of G₁-to-S-phase transition, was consequently hypophosphorylated, while GATA-1, master transcription factor in erythropoiesis was reduced. The resultant decreased expression of cyclin A and D2 retarded cell-cycle progression in erythroid cells and the K562 cell line. As a second major effect, HZ and HNE inhibited protein expression of crucial receptors (R): transferrinR1, stem cell factorR, interleukin-3R, and erythropoietinR. The reduced receptor expression and the impaired cell-cycle activity decreased the production of cells expressing glycophorin-A and hemoglobin. Present data confirm the inhibitory role of HZ, identify HNE as one HZ-generated inhibitory molecule and describe molecular targets of HNE in erythroid progenitors possibly involved in erythropoiesis inhibition in malaria anemia.

Role of the Lipoperoxidation Product 4-Hydroxynonenal in the Pathogenesis of Severe Malaria Anemia and Malaria Immunodepression

Oxidative stress plays an important role in the pathogenesis of falciparum malaria, a disease still claiming close to 1 million deaths and 200 million new cases per year. Most frequent complications are severe anemia, cerebral malaria, and immunodepression, the latter being constantly present in all forms of malaria. Complications are associated with oxidative stress and lipoperoxidation. Its final product 4-hydroxynonenal (4-HNE), a stable yet very reactive and diffusible molecule, forms covalent conjugates with proteins, DNA, and phospholipids and modulates important cell functions at very low concentrations. Since oxidative stress plays important roles in the pathogenesis of severe malaria, it appears important to explore the role of 4-HNE in two important malaria complications such as malaria anemia and malaria immunodepression where oxidative stress is considered to be involved. In this review we will summarize data about 4-HNE chemistry, its biologically relevant chemical properties, and its role as regulator of physiologic processes and as pathogenic factor. We will review studies documenting the role of 4-HNE in severe malaria with emphasis on malaria anemia and immunodepression. Data from other diseases qualify 4-HNE both as oxidative stress marker and as pathomechanistically important molecule. Further studies are needed to establish 4-HNE as accepted pathogenic factor in severe malaria.

Malarial pigment haemozoin, IFN-gamma, TNF-alpha, IL-1beta and LPS do not stimulate expression of inducible nitric oxide synthase and production of nitric oxide in immuno-purified human monocytes

BackgroundEnhanced production of nitric oxide (NO) following upmodulation of the inducible isoform of NO synthase (iNOS) by haemozoin (HZ), inflammatory cytokines and LPS may provide protection against Plasmodium falciparum malaria by killing hepatic and blood forms of parasites and inhibiting the cytoadherence of parasitized erythrocytes (RBC) to endothelial cells. Monocytes and macrophages are considered to contribute importantly to protective upregulation of iNOS and production of NO. Data obtained with murine phagocytes fed with human HZ and synthetic HZ (sHZ) indicate that supplemental treatment of those cells with IFN-gamma elicited significant increases in protein and mRNA expression of iNOS and NO production, providing a potential mechanism linking HZ phagocytosis and increased production of NO. Purpose of this study was to analyse the effect of P. falciparum HZ and sHZ supplemental to treatment with IFN-gamma and/or a stimulatory cytokine-LPS mix on iNOS protein and mRNA expression in immuno-purified human monocytes.MethodsAdherent immunopurified human monocytes (purity >85%), and murine phagocytic cell lines RAW 264.7, N11 and ANA1 were fed or not with P. falciparum HZ or sHZ and treated or not with IFN-gamma or a stimulatory cytokine-LPS mix. Production of NO was quantified in supernatants, iNOS protein and mRNA expression were measured after immunoprecipitation and Western blotting and quantitative RT-PCT, respectively.ResultsPhagocytosis of HZ/sHZ by human monocytes did not increase iNOS protein and mRNA expression and NO production either after stimulation by IFN-gamma or the cytokine-LPS mix. By contrast, in HZ/sHZ-laden murine macrophages, identical treatment with IFN-gamma and the cytokine-LPS mix elicited significant increases in protein and mRNA expression of iNOS and NOS metabolites production, in agreement with literature data.ConclusionResults indicate that human monocytes fed or not with HZ/sHZ were constantly unable to express iNOS and generate NOS metabolites even after stimulation with IFN-gamma or a cytokine-LSP mix that were very active on HZ-fed murine phagocytic lines. Present data do not support the hypothesis that monocytes are mediators of anti-parasitic defence in clinical malaria via activation of iNOS and production of NO, and suggest caution in extrapolating data obtained with murine or hybrid systems to human malaria.

Transfer of 4-hydroxynonenal from parasitized to non-parasitized erythrocytes in rosettes. Proposed role in severe malaria anemia

Severe anaemia is a life‐threatening complication of falciparum malaria associated with loss of predominantly non‐parasitized red blood cells (npRBCs). This poorly elucidated process might be influenced by (i) rosettes, i.e. npRBCs cytoadherent to haemozoin‐containing parasitized RBCs (pRBCs) and (ii) generation in pRBCs of 4‐hydroxynonenal (4‐HNE) through haemozoin‐catalysed lipid peroxidation. We explored whether close proximity in rosettes may facilitate 4‐HNE transfer to npRBCs, which is likely to enhance their phagocytosis and contribute to malaria anaemia. Fluorescence microscopy and flow cytometry data indicated 4‐HNE transfer to npRBCs in rosettes. Rosettes were formed by 64·8 ± 1·8% varO‐expressing pRBCs, and 8·7 ± 1·1% npRBCs were positive for 4‐HNE‐protein‐conjugates, while low‐rosetting parasites generated only 2·4 ± 1·1% 4‐HNE‐conjugate‐positive npRBCs. 4‐HNE transfer decreased after blocking rosetting by monoclonal antibodies. A positive linear relationship between rosette frequency and 4‐HNE‐conjugates in npRBCs was found in 40 malaria patients, a first indication for a role of rosetting in npRBCs modifications in vivo. Children with severe malaria anaemia had significantly higher percentages of 4‐HNE‐conjugate‐positive npRBCs compared to children with uncomplicated malaria. In conclusion, 4‐HNE transfer from pRBCs to npRBCs in rosettes is suggested to play a role in the phagocytic removal of large numbers of npRBCs, the hallmark of severe malaria anaemia.

Blood oxidative stress markers and Plasmodium falciparum malaria in non-immune African children.

Converging in vitro evidence and clinical data indicate that oxidative stress may play important roles in Plasmodium falciparum malaria, notably in the pathogenesis of severe anaemia. However, oxidative modifications of the red blood cell (RBC)‐membrane by 4‐hydroxynonenal (4‐HNE) and haemoglobin‐binding, previously hypothesized to contribute mechanistically to the pathogenesis of clinical malaria, have not yet been tested for clinical significance. In 349 non‐immune Mozambican newborns recruited in a double‐blind placebo‐controlled chemoprophylaxis trial, oxidative markers including 4‐HNE‐conjugates and membrane‐bound haemoglobin were longitudinally assessed from 2·5 to 24 months of age, at first acute malaria episode and in convalescence. During acute malaria, 4‐HNE‐conjugates were shown to increase significantly in parasitized and non‐parasitized RBCs. In parallel, advanced oxidation protein products (AOPP) rose in plasma. 4‐HNE‐conjugates correlated with AOPP and established plasma but not with RBC oxidative markers. High individual levels of 4‐HNE‐conjugates were predictive for increased malaria incidence rates in children until 2 years of life and elevated 4‐HNE‐conjugates in convalescence accompanied sustained anaemia after a malaria episode, indicating 4‐HNE‐conjugates as a novel patho‐mechanistic factor in malaria. A second oxidative marker, haemoglobin binding to RBC‐membranes, hypothesized to induce clearing of RBCs from circulation, was predictive for lower malaria incidence rates. Further studies will show whether or not higher membrane‐haemoglobin values at the first malaria episode may provide protection against malaria.

Simultaneous determination of phagocytosis of Plasmodium falciparum -parasitized and non-parasitized red blood cells by flow cytometry

BackgroundSevere falciparum malaria anaemia (SMA) is a frequent cause of mortality in children and pregnant women. The most important determinant of SMA appears to be the loss of non-parasitized red blood cells (np-RBCs) in excess of loss of parasitized (p-) RBCs at schizogony. Based on data from acute SMA where excretion of haemoglobin in urine and increased plasma haemoglobin represented respectively less than 1% and 0.5% of total Hb loss, phagocytosis appears to be the predominant mechanism of removal of np- and p-RBC.Estimates indicate that np-RBCs are cleared in approximately 10-fold excess compared to p-RBCs. An even larger removal of np-RBCs has been described in vivax malaria anaemia. Estimates were based on two single studies both performed on neurosyphilitic patients who underwent malaria therapy. As the share of np-RBC removal is likely to vary between wide limits, it is important to assess the contribution of both np- and p-RBC populations to overall RBC loss, and disclose the mechanism of such variability. As available methods do not discriminate between the removal of np- vs p-RBCs, the purpose of this study was to set up a system allowing the simultaneous determination of phagocytosis of p- and np-RBC in the same sample.Methods and ResultsPhagocytosis of p- and np-RBCs was quantified in the same sample using double-labelled target cells and the human phagocytic cell-line THP-1, pre-activated by TNF and IFNγ to enhance their phagocytic activity. Target RBCs were double-labelled with fluorescent carboxyfluorescein-succinimidyl ester (CF-SE) and the DNA label ethidium bromide (EB). EB, a DNA label, allowed to discriminate p-RBCs that contain parasitic DNA from the np-RBCs devoid of DNA. FACS analysis of THP-1 cells fed with double-labelled RBCs showed that p- and np-RBCs were phagocytosed in different proportions in relation to parasitaemia.ConclusionsThe assay allowed the analysis of phagocytosis rapidly and with low subjective error, and the differentiation between phagocytosed p- and np-RBCs in the same sample. The presented method may help to analyse the factors or conditions that modulate the share of np-RBC removal in vitro and in vivo and lead to a better understanding of the pathogenesis of SMA.

Oxidative stress-mediated antimalarial activity of plakortin, a natural endoperoxide from the tropical sponge Plakortis simplex.

Plakortin, a polyketide endoperoxide from the sponge Plakortis simplex has antiparasitic activity against P. falciparum. Similar to artemisinin, its activity depends on the peroxide functionality. Plakortin induced stage-, dose- and time-dependent morphologic anomalies, early maturation delay, ROS generation and lipid peroxidation in the parasite. Ring damage by 1 and 10 µM plakortin led to parasite death before schizogony at 20 and 95%, respectively. Treatment of late schizonts with 1, 2, 5 and 10 µM plakortin resulted in decreased reinfection rates by 30, 50, 61 and 65%, respectively. In both rings and trophozoites, plakortin induced a dose- and time-dependent ROS production as well as a significant lipid peroxidation and up to 4-fold increase of the lipoperoxide breakdown product 4-hydroxynonenal (4-HNE). Antioxidants and the free radical scavengers trolox and N-acetylcysteine significantly attenuated the parasite damage. Plakortin generated 4-HNE conjugates with the P. falciparum proteins: heat shock protein Hsp70-1, endoplasmatic reticulum-standing Hsp70-2 (BiP analogue), V-type proton ATPase catalytic subunit A, enolase, the putative vacuolar protein sorting-associated protein 11, and the dynein heavy chain-like protein, whose specific binding sites were identified by mass spectrometry. These proteins are crucially involved in protein trafficking, transmembrane and vesicular transport and parasite survival. We hypothesize that binding of 4-HNE to functionally relevant parasite proteins may explain the observed plakortin-induced morphologic aberrations and parasite death. The identification of 4-HNE-protein conjugates may generate a novel paradigm to explain the mechanism of action of pro-oxidant, peroxide-based antimalarials such as plakortin, artemisinins and synthetic endoperoxides.

Single-stranded DNA fragments of insect-specific nuclear polyhedrosis virus act as selective DNA insecticides for gypsy moth control.

This paper focuses on the DNA insecticides as a novel preparation against gypsy moth (Lymantria dispar) based on DNA fragments of the anti-apoptotic gene of its nuclear polyhedrosis virus. It was found that the external application of a solution with two single-stranded DNA fragments from BIR and RING domains of LdMNPV (L.dispar multicapsid nuclear polyhedrosis virus) IAP-3 (inhibitor of apoptosis) gene induces a significantly higher mortality of gypsy moth caterpillars in comparison with the application of the control solutions. This effect does not depend on the infection of caterpillars with LdMNPV. The results also show that DNA insecticides based on LdMNPV IAP-3 gene fragments can be selective in action, and at least are not harmful to tobacco hornworm (Manduca sexta) and black cutworm (Agrotis ipsilon). Part of the gypsy moth genome cloned with the fragments of BIR and RING domains of LdMNPV IAP-3 gene as primers, has an overlap with the corresponding part of the LdMNPV IAP-3 gene and L.dispar IAP-1 mRNA for an inhibitor of apoptosis protein with the high cover by query, allows assuming that we cloned a part of gypsy moth anti-apoptosis gene. This finding gives the grounding that proposed here DNA insecticides might act through the blocking of the mechanisms involved in post transcriptional expression of insect anti-apoptosis genes. The results show the insecticidal potential of the viral genome fragments that can be used to create safe and relatively fast-acting DNA insecticides to control the quantity of gypsy moth populations, important task for forestry and agriculture.