Post-Translational Modifications of Proteins of Malaria Parasites during the Life Cycle.

Post-translational modifications (PTMs) are essential for regulating protein functions, influencing various fundamental processes in eukaryotes. These include, but are not limited to, cell signaling, protein trafficking, the epigenetic control of gene expression, and control of the cell cycle, as well as cell proliferation, differentiation, and interactions between cells. In this review, we discuss protein PTMs that play a key role in the malaria parasite biology and its pathogenesis. Phosphorylation, acetylation, methylation, lipidation and lipoxidation, glycosylation, ubiquitination and sumoylation, nitrosylation and glutathionylation, all of which occur in malarial parasites, are reviewed. We provide information regarding the biological significance of these modifications along all phases of the complex life cycle of Plasmodium spp. Importantly, not only the parasite, but also the host and vector protein PTMs are often crucial for parasite growth and development. In addition to metabolic regulations, protein PTMs can result in epitopes that are able to elicit both innate and adaptive immune responses of the host or vector. We discuss some existing and prospective results from antimalarial drug discovery trials that target various PTM-related processes in the parasite or host.

Posttranslational Modification of Human Cytochrome CYP4F11 by 4-Hydroxynonenal Impairs ω-Hydroxylation in Malaria Pigment Hemozoin-Fed Monocytes: The Role in Malaria Immunosuppression.

Malaria is a frequent parasitic infection becomes life threatening due to the disequilibrated immune responses of the host. Avid phagocytosis of malarial pigment hemozoin (HZ) and HZ-containing Plasmodium parasites incapacitates monocyte functions by bioactive lipoperoxidation products 4-hydroxynonenal (4-HNE) and hydroxyeicosatetraenoic acids (HETEs). CYP4F conjugation with 4-HNE is hypothesised to inhibit ω-hydroxylation of 15-HETE, leading to sustained monocyte dysfunction caused by 15-HETE accumulation. A combined immunochemical and mass-spectrometric approach identified 4-HNE-conjugated CYP4F11 in primary human HZ-laden and 4-HNE-treated monocytes. Six distinct 4-HNE-modified amino acid residues were revealed, of which C260 and H261 are localized in the substrate recognition site of CYP4F11. Functional consequences of enzyme modification were investigated on purified human CYP4F11. Palmitic acid, arachidonic acid, 12-HETE, and 15-HETE bound to unconjugated CYP4F11 with apparent dissociation constants of 52, 98, 38, and 73 µM, respectively, while in vitro conjugation with 4-HNE completely blocked substrate binding and enzymatic activity of CYP4F11. Gas chromatographic product profiles confirmed that unmodified CYP4F11 catalysed the ω-hydroxylation while 4-HNE-conjugated CYP4F11 did not. The 15-HETE dose dependently recapitulated the inhibition of the oxidative burst and dendritic cell differentiation by HZ. The inhibition of CYP4F11 by 4-HNE with consequent accumulation of 15-HETE is supposed to be a crucial step in immune suppression in monocytes and immune imbalance in malaria.

An Optimized Dihydrodibenzothiazepine Lead Compound (SBI-0797750) as a Potent and Selective Inhibitor of Plasmodium falciparum and P. vivax Glucose 6-Phosphate Dehydrogenase 6-Phosphogluconolactonase.

In Plasmodium, the first two and rate-limiting enzymes of the pentose phosphate pathway, glucose 6-phosphate dehydrogenase (G6PD) and the 6-phosphogluconolactonase, are bifunctionally fused to a unique enzyme named GluPho, differing structurally and mechanistically from the respective human orthologs. Consistent with the enzyme's essentiality for malaria parasite proliferation and propagation, human G6PD deficiency has immense impact on protection against severe malaria, making PfGluPho an attractive antimalarial drug target. Herein we report on the optimized lead compound N-(((2R,4S)-1-cyclobutyl-4-hydroxypyrrolidin-2-yl)methyl)-6-fluoro-4-methyl-11-oxo-10,11-dihydrodibenzo[b,f][1,4]thiazepine-8-carboxamide (SBI-0797750), a potent and fully selective PfGluPho inhibitor with robust nanomolar activity against recombinant PfGluPho, PvG6PD, and P. falciparum blood-stage parasites. Mode-of-action studies have confirmed that SBI-0797750 disturbs the cytosolic glutathione-dependent redox potential, as well as the cytosolic and mitochondrial H2O2 homeostasis of P. falciparum blood stages, at low nanomolar concentrations. Moreover, SBI-0797750 does not harm red blood cell (RBC) integrity and phagocytosis and thus does not promote anemia. SBI-0797750 is therefore a very promising antimalarial lead compound.

Relaxometric studies of erythrocyte suspensions infected by Plasmodium falciparum: a tool for staging infection and testing anti-malarial drugs.

PURPOSE: Malaria is a global health problem with the most malignant form caused by Plasmodium falciparum (P. falciparum). Parasite maturation in red blood cells (RBCs) is accompanied by changes including the formation of paramagnetic hemozoin (HZ) nanocrystals, and increased metabolism and variation in membrane lipid composition. Herein, MR relaxometry (MRR) was applied to investigate water exchange across RBCs' membrane and HZ formation in parasitized RBCs. METHODS: Transverse water protons relaxation rate constants (R2 = 1/T2 ) were measured for assessing HZ formation in P. falciparum-parasitized human RBCs. Moreover, water exchange lifetimes across the RBC membrane (τi ) were assessed by measuring longitudinal relaxation rate constants (R1 = 1/T1 ) at 21.5 MHz in the presence of a gadolinium complex dissolved in the suspension medium. RESULTS: τi increased after invasion of parasites (ring stage, mean τi / τi0 = 1.234 ± 0.022) and decreased during maturation to late trophozoite (mean τi / τi0 = 0.960 ± 0.075) and schizont stages (mean τi / τi0 = 1.019 ± 0.065). The HZ accumulation in advanced stages was revealed by T2 -shortening. The curves reporting R2 (1/T2 ) vs. magnetic field showed different slopes for non-parasitized RBCs (npRBCs) and parasitized RBCs (pRBCs), namely 0.003 ± 0.001 for npRBCs, 0.009 ± 0.002, 0.028 ± 0.004 and 0.055 ± 0.002 for pRBCs at ring-, early trophozoite-, and late trophozoite stage, respectively. Antimalarial molecules dihydroartemisinin and chloroquine elicited measurable changes in parasitized RBCs, namely dihydroartemisinin modified τi , whereas the interference of chloroquine with HZ formation was detectable by a significant T2 increase. CONCLUSIONS: MRR can be considered a useful tool for reporting on P. falciparum blood stages and for screening potential antimalarial molecules.

Syk inhibitors interfere with erythrocyte membrane modification during P falciparum growth and suppress parasite egress.

Band 3 (also known as the anion exchanger, SLCA1, AE1) constitutes the major attachment site of the spectrin-based cytoskeleton to the erythrocyte's lipid bilayer and thereby contributes critically to the stability of the red cell membrane. During the intraerythrocytic stage of Plasmodium falciparum's lifecycle, band 3 becomes tyrosine phosphorylated in response to oxidative stress, leading to a decrease in its affinity for the spectrin/actin cytoskeleton and causing global membrane destabilization. Because this membrane weakening is hypothesized to facilitate parasite egress and the consequent dissemination of released merozoites throughout the bloodstream, we decided to explore which tyrosine kinase inhibitors might block the kinase-induced membrane destabilization. We demonstrate here that multiple Syk kinase inhibitors both prevent parasite-induced band 3 tyrosine phosphorylation and inhibit parasite-promoted membrane destabilization. We also show that the same Syk kinase inhibitors suppress merozoite egress near the end of the parasite's intraerythrocytic lifecycle. Because the entrapped merozoites die when prevented from escaping their host erythrocytes and because some Syk inhibitors have displayed long-term safety in human clinical trials, we suggest Syk kinase inhibitors constitute a promising class of antimalarial drugs that can suppress parasitemia by inhibiting a host target that cannot be mutated by the parasite to evolve drug resistance.

Malaria-derived hemozoin exerts early modulatory effects on the phenotype and maturation of human dendritic cells.

Plasmodium falciparum (P. falciparum)-induced effects on the phenotype of human dendritic cells (DC) could contribute to poor induction of long-lasting protective immunity against malaria. DC ability to present antigens to naïve T cells, thus initiating adaptive immune responses depends on complex switches in chemokine receptors, production of soluble mediators and expression of molecules enabling antigen-presentation and maturation. To examine the cellular basis of these processes in the context of malaria, we performed detailed analysis of early events following exposure of human monocyte-derived DC to natural hemozoin (nHZ) and the synthetic analog of its heme core, ß-hematin. DC exposed to either molecule produced high levels of the inflammatory chemokine MCP-1, showed continuous high expression of the inflammatory chemokine receptor CCR5, no upregulation of the lymphoid homing receptor CCR7 and no cytoskeletal actin redistribution with loss of podosomes. DC partially matured as indicated by increased expression of major histocompatibility complex (MHC) class II and CD86 following nHZ and ß-hematin exposure, however there was a lack in expression of the maturation marker CD83 following nHZ but not ß-hematin exposure. Overall our data demonstrate that exposure to nHZ partially impairs the capacity of DC to mature, an effect in part differential to ß-hematin.

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.

Plasma advanced oxidative protein products are associated with anti-oxidative stress pathway genes and malaria in a longitudinal cohort.

BACKGROUND: Advanced oxidation protein products (AOPP) are newly identified efficient oxidative stress biomarkers. In a longitudinal birth cohort the effects were investigated of genetic polymorphisms in five oxidative pathway genes on AOPP levels. METHODS: This study is part of a three-arm randomized, double-blind, placebo-controlled trial. Three hundred and twelve children were included in the present study with AOPP levels measured at 2.5, 5.5, 10.5, 15 and 24 months of age. Twelve polymorphisms were genotyped in five oxidative stress pathway genes: glutathione reductase (GSR), glutamylcysteine synthetase (GCLC), glutathione S-transferase (GST) P1, haem oxygenase 1 (HMOX1) and superoxide dismutase 2 (SOD2) in 298 children. There were 284 children assessed for anaemia and clinical malaria infection at the age of 24 months. RESULTS: Two principal components (PCA1 and PCA2) were derived from the AOPP levels measured at the five time points. PCA1 was significantly associated with anaemia (p = 0.0002), and PCA2 with clinical malaria infection (p = 0.047). In the K-Means Cluster Analysis based on levels of AOPP, children were clustered into two groups: Group A (lower AOPP levels) and Group B (higher AOPP levels). The cluster membership was significantly associated with anaemia (p =0.003) as well as with the GSR RS3594 polymorphism (p = 0.037). Mixed linear regression analyses found that the single nucleotide polymorphisms GCLC RS10948751 and HMOX1 RS17885925 were significantly associated with AOPP levels (p = 0.030 and p = 0.027, respectively). CONCLUSION: Plasma AOPP levels were predictive for anaemia and oxidative stress markers for clinical malaria infection in two year old children. Several polymorphisms in GCLC, GSR and HMOX1 genes were associated with oxidative stress status of these children.

Hemozoin induces lung inflammation and correlates with malaria-associated acute respiratory distress syndrome.

Malaria-associated acute respiratory distress syndrome (MA-ARDS) is a deadly complication of malaria, and its pathophysiology is insufficiently understood. Both in humans and in murine models, MA-ARDS is characterized by marked pulmonary inflammation. We investigated the role of hemozoin in MA-ARDS in C57Bl/6 mice infected with Plasmodium berghei NK65, P. berghei ANKA, and P. chabaudi AS. By quantifying hemozoin in the lungs and measuring the disease parameters of MA-ARDS, we demonstrated a highly significant correlation between pulmonary hemozoin concentrations, lung weights, and alveolar edema. Histological analysis of the lungs demonstrated that hemozoin is localized in phagocytes and infected erythrocytes, and only occasionally in granulocytes. Species-specific differences in hemozoin production, as measured among individual schizonts, were associated with variations in pulmonary pathogenicity. Furthermore, both pulmonary hemozoin and lung pathology were correlated with the number of infiltrating inflammatory cells, an increased pulmonary expression of cytokines, chemokines, and enzymes, and concentrations of alveolar vascular endothelial growth factor. The causal relationship between hemozoin and inflammation was investigated by injecting P. falciparum-derived hemozoin intravenously into malaria-free mice. Hemozoin potently induced the pulmonary expression of proinflammatory chemokines (interferon-γ inducible protein-10/CXC-chemokine ligand (CXCL)10, monocyte chemotactic protein-1/CC-chemokine ligand 2, and keratinocyte-derived chemokine/CXCL1), cytokines (IL-1ß, IL-6, IL-10, TNF, and transforming growth factor-ß), and other inflammatory mediators (inducible nitric oxide synthase, heme oxygenase-1, nicotinamide adenine dinucleotide phosphate- oxidase-2, and intercellular adhesion molecule-1). Thus, hemozoin correlates with MA-ARDS and induces pulmonary inflammation.

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.

Micromolar Dihydroartemisinin Concentrations Elicit Lipoperoxidation in Plasmodium falciparum-Infected Erythrocytes.

Malaria is still the most important parasitic infectious disease. Numerous substances are known to have antimalarial activity; among them, artemisinin is the most widely used one, and artemisinin-based combination therapy (ACT) is recommended for the treatment of Plasmodium falciparum (P.f.) malaria. Antitumor, immunomodulatory, and other therapeutic applications of artemisinin are under extensive study. Several different mechanisms of action were proposed for dihydroartemisinin (DHA), the active metabolite of artemisinin, such as eliciting oxidative stress in target cells. The goal of this study is to monitor the generation of reactive oxygen species (ROS) and lipid peroxidation product 4-hydroxynonenal (4-HNE) by DHA in P.f.-infected human erythrocytes. Checking ROS and 4-HNE-protein adducts kinetics along the maturation of the parasite, we detected the highest level of 4-HNE in ring forms of P.f. due to DHA treatment. Low micromolar concentrations of DHA quickly induced levels of 4-HNE-adducts which are supposed to be damaging. Mass spectrometry identified the P.f. protein cysteine proteinase falcipain-1 as being heavily modified by 4-HNE, and plausibly, 4-HNE conjugation with vital P.f. proteins might contribute to DHA-elicited parasite death. In conclusion, significant 4-HNE accumulation was detectable after DHA treatment, though, at concentrations well above pharmacologically effective ranges in malaria treatment, but at concentrations described for antitumor activity. Thus, lipid peroxidation with consequent 4-HNE conjugation of functionally relevant proteins might be considered as a uniform mechanism for how DHA potentiates antimalarials' action in ACT and controls the progression of tumors.

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.

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.

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

BACKGROUND: Severe 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 RESULTS: Phagocytosis 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. CONCLUSIONS: The 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.

Malaria Pigment Hemozoin Impairs GM-CSF Receptor Expression and Function by 4-Hydroxynonenal.

Malarial pigment hemozoin (HZ) generates the lipoperoxidation product 4-hydroxynonenal (4-HNE), which is known to cause dysregulation of the immune response in malaria. The inhibition of granulocyte macrophage colony-stimulating factor (GM-CSF)-dependent differentiation of dendritic cells (DC) by HZ and 4-HNE was previously described in vitro, and the GM-CSF receptor (GM-CSF R) was hypothesised to be a primary target of 4-HNE in monocytes. In this study, we show the functional impact of HZ on GM-CSF R in monocytes and monocyte-derived DC by (i) impairing GM-CSF binding by 50 ± 9% and 65 ± 14%, respectively (n = 3 for both cell types); (ii) decreasing the expression of GM-CSF R functional subunit (CD116) on monocyte's surface by 36 ± 11% (n = 6) and in cell lysate by 58 ± 16% (n = 3); and (iii) binding of 4-HNE to distinct amino acid residues on CD116. The data suggest that defective DC differentiation in malaria is caused by GM-CSF R dysregulation and GM-CSF R modification by lipoperoxidation product 4-HNE via direct interaction with its CD116 subunit.

Involvement of inflammatory chemokines in survival of human monocytes fed with malarial pigment.

Hemozoin (HZ)-fed monocytes are exposed to strong oxidative stress, releasing large amounts of peroxidation derivatives with subsequent impairment of numerous functions and overproduction of proinflammatory cytokines. However, the histopathology at autopsy of tissues from patients with severe malaria showed abundant HZ in Kupffer cells and other tissue macrophages, suggesting that functional impairment and cytokine production are not accompanied by cell death. The aim of the present study was to clarify the role of HZ in cell survival, focusing on the qualitative and temporal expression patterns of proinflammatory and antiapoptotic molecules. Immunocytochemical and flow cytometric analyses showed that the long-term viability of human monocytes was unaffected by HZ. Short-term analysis by macroarray of a complete panel of cytokines and real-time reverse transcription (RT)-PCR experiments showed that HZ immediately induced interleukin-1ß (IL-1ß) gene expression, followed by transcription of eight additional chemokines (IL-8, epithelial cell-derived neutrophil-activating peptide 78 [ENA-78], growth-regulated oncogene α [GROα], GROß, GROγ, macrophage inflammatory protein 1α [MIP-1α], MIP-1ß, and monocyte chemoattractant protein 1 [MCP-1]), two cytokines (tumor necrosis factor alpha [TNF-α] and IL-1receptor antagonist [IL-1RA]), and the cytokine/chemokine-related proteolytic enzyme matrix metalloproteinase 9 (MMP-9). Furthermore, real-time RT-PCR showed that 15-HETE, a potent lipoperoxidation derivative generated by HZ through heme catalysis, recapitulated the effects of HZ on the expression of four of the chemokines. Intermediate-term investigation by Western blotting showed that HZ increased expression of HSP27, a chemokine-related protein with antiapoptotic properties. Taken together, the present data suggest that apoptosis of HZ-fed monocytes is prevented through a cascade involving 15-HETE-mediated upregulation of IL-1ß transcription, rapidly sustained by chemokine, TNF-α, MMP-9, and IL-1RA transcription and upregulation of HSP27 protein expression.

Plasmodium Impairs Antibacterial Innate Immunity to Systemic Infections in Part Through Hemozoin-Bound Bioactive Molecules.

One complication of malaria is increased susceptibility to invasive bacterial infections. Plasmodium infections impair host immunity to non-Typhoid Salmonella (NTS) through heme-oxygenase I (HO-I)-induced release of immature granulocytes and myeloid cell-derived IL-10. Yet, it is not known if these mechanisms are specific to NTS. We show here, that Plasmodium yoelii 17XNL (Py) infected mice had impaired clearance of systemic Listeria monocytogenes (Lm) during both acute parasitemia and up to 2 months after clearance of Py infected red blood cells that was independent of HO-I and IL-10. Py-infected mice were also susceptible to Streptococcus pneumoniae (Sp) bacteremia, a common malaria-bacteria co-infection, with higher blood and spleen bacterial burdens and decreased survival compared to naïve mice. Mechanistically, impaired immunity to Sp was independent of HO-I, but was dependent on Py-induced IL-10. Splenic phagocytes from Py infected mice exhibit an impaired ability to restrict growth of intracellular Lm, and neutrophils from Py-infected mice produce less reactive oxygen species (ROS) in response to Lm or Sp. Analysis also identified a defect in a serum component in Py-infected mice that contributes to reduced production of ROS in response to Sp. Finally, treating naïve mice with Plasmodium-derived hemozoin containing naturally bound bioactive molecules, excluding DNA, impaired clearance of Lm. Collectively, we have demonstrated that Plasmodium infection impairs host immunity to diverse bacteria, including S. pneumoniae, through multiple effects on innate immunity, and that a parasite-specific factor (Hz+bound bioactive molecules) directly contributes to Plasmodium-induced suppression of antibacterial innate immunity.

Oxidative Stress, Lipid Peroxidation, and Loss of Hyaluronic Acid in the Human Vitreous Affected by Synchysis Scintillans.

The aim of this study was to assess the oxidative stress status in eyes affected by synchysis scintillans and to compare it to vitreoretinal disorders without synchysis scintillans. Human aqueous and vitreous humors were obtained during vitrectomy from thirty-seven otherwise healthy patients that were randomly chosen among patients that had to undergo a 25-gauge pars plana vitrectomy from the central vitreous cavity, for either synchysis scintillans (n = 16) or vitreoretinal disorders without synchysis scintillans (n = 21), such as idiopathic epimacular membrane (n = 12), macular hole (n = 5), or rhegmatogenous retinal detachment (n = 4). The redox parameters thiobarbituric acid reactive substances (TBARS), a measurement of lipid peroxidation, nitrite concentration, an estimate of nitric oxide (NO) production, 4-hydroxynonenal (4-HNE)-protein conjugates, a structural protein modification by lipid peroxidation product 4-HNE, and the antioxidative activities of Cu,Zn-superoxide dismutase (SOD), and catalase were measured in aqueous and vitreous humors and compared between synchysis scintillans affected and not-affected patients. TBARS and nitrite levels of the vitreous humor were significantly higher in patients with synchysis scintillans as compared to patients affected by vitreoretinal disorders without synchysis scintillans. Synchysis scintillans patients had significantly lower activities of SOD and catalase both in aqueous and vitreous humors than patients with vitreoretinal disorders without synchysis. The consequently higher lipoperoxide-dependent 4-HNE production in synchysis scintillans was detectable in aqueous and vitreous humors as a significant increased accumulation of 4-HNE-protein conjugates vs nonsynchysis vitreoretinal disorders. Additionally, hyaluronic acid (HA) was significantly decreased in the vitreous body of synchysis scintillans patients. The data consistently show that synchisis scintillans is accompanied by a redox imbalance with increased oxidative modifications of 4-HNE proteins and loss of HA, both of likely importance for remote damages of the retina. It remains to be proven whether a therapeutic strategy which targets oxidative stress may be effective in the treatment of synchysis patients.

Neutrophil extracellular traps drive inflammatory pathogenesis in malaria.

Neutrophils are essential innate immune cells that extrude chromatin in the form of neutrophil extracellular traps (NETs) when they die. This form of cell death has potent immunostimulatory activity. We show that heme-induced NETs are essential for malaria pathogenesis. Using patient samples and a mouse model, we define two mechanisms of NET-mediated inflammation of the vasculature: activation of emergency granulopoiesis via granulocyte colony-stimulating factor production and induction of the endothelial cytoadhesion receptor intercellular adhesion molecule-1. Soluble NET components facilitate parasite sequestration and mediate tissue destruction. We demonstrate that neutrophils have a key role in malaria immunopathology and propose inhibition of NETs as a treatment strategy in vascular infections.