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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.

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 Pf GluPho 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 Pf GluPho inhibitor with robust nanomolar activity against recombinant Pf GluPho, Pv G6PD, 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 H 2 O 2 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.

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., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Plasmodium falciparum digests up to 75% of erythrocyte (red blood cell [RBC]) hemoglobin and forms hemozoin. Phagocytosed hemozoin and trophozoites inhibit important monocyte functions. Delipidized trophozoites and hemozoin were remarkably less toxic to monocytes. Parasitized RBCs and hemozoin contained large amounts of mostly esterified monohydroxy derivatives (OH-PUFAs), the stable end products of peroxidation of polyenoic fatty acids. The concentrations of OH-PUFA were 1.8 micromoles per liter RBCs in nonparasitized RBCs, 11.1 micromoles per liter RBCs in rings, 35 micromoles per liter RBCs in trophozoites; and approximately 90 micromoles per liter RBC equivalents in hemozoin. In parasitized RBCs and hemozoin a complex mixture of monohydroxy derivatives of arachidonic (HETEs) and linoleic (HODEs) acid was determined. Respectively, 13- and 9-HODE and 9- and 12-HETE were predominant in hemozoin and parasitized RBCs. The estimated concentrations of all HETE isomers were 33 and 39 micromoles per liter RBCs or RBC equivalents in trophozoites and hemozoin, respectively. No evidence of lipoxygenase activity was found, whereas the large number of positional and optical isomers, the racemic structure, and their generation by incubation of arachidonic acid with hemozoin indicated nonenzymatic origin via heme-catalysis. Sub/low micromolar concentrations of 12- and 15-HETE were toxic to monocytes, whereas HODE isomers were ineffective. Low micromolar concentrations of HETE isomers were estimated to be similarly present in monocytes after phagocytosis of trophozoites or hemozoin. Thus, specific products of heme-catalyzed lipid peroxidation appear to contribute to hemozoin toxicity to phagocytes and may thus play a role in increased cytoadherence, vascular permeability, and chemotaxis, as well as in immunodepression in malaria.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Recently matrix metalloproteinase-9 (MMP-9) and its endogenous inhibitor (tissue inhibitor of metalloproteinase-1, TIMP-1) have been implicated in complicated malaria. In vivo, mice with cerebral malaria (CM) display high levels of both MMP-9 and TIMP-1, and in human patients TIMP-1 serum levels directly correlate with disease severity. In vitro, natural haemozoin (nHZ, malarial pigment) enhances monocyte MMP-9 expression and release. The present study analyses the effects of nHZ on TIMP-1 regulation in human adherent monocytes. nHZ induced TIMP-1 mRNA expression and protein release, and promoted TNF-α, IL-1β, and MIP-1α/CCL3 production. Blocking antibodies or recombinant cytokines abrogated or mimicked nHZ effects on TIMP-1, respectively. p38 MAPK and NF-κB inhibitors blocked all nHZ effects on TIMP-1 and pro-inflammatory molecules. Still, total gelatinolytic activity was enhanced by nHZ despite TIMP-1 induction. Collectively, these data indicate that nHZ induces inflammation-mediated expression and release of human monocyte TIMP-1 through p38 MAPK- and NF-κB-dependent mechanisms. However, TIMP-1 induction is not sufficient to counterbalance nHZ-dependent MMP-9 enhancement. Future investigation on proteinase-independent functions of TIMP-1 (i.e. cell survival promotion and growth/differentiation inhibition) is needed to clarify the role of TIMP-1 in malaria pathogenesis. [ABSTRACT FROM AUTHOR]

Abstract: Objective: To investigate the role of macrophage inflammatory protein-1alpha (MIP-1alpha) in the detrimental enhancement of matrix metalloproteinase-9 (MMP-9) expression, release and activity induced by phagocytosis of malarial pigment (haemozoin, HZ) in human monocytes. Methods: Human adherent monocytes were unfed/fed with native HZ for 2 h. After 24 hours, MIP-1alpha production was evaluated by ELISA in cell supernatants. Alternatively, HZ-unfed/fed monocytes were treated in presence/absence of anti-human MIP-1alpha blocking antibodies or recombinant human MIP-1alpha for 15 h (RNA studies) or 24 h (protein studies); therefore, MMP-9 mRNA expression was evaluated in cell lysates by Real Time RT-PCR, whereas proMMP-9 and active MMP-9 protein release were measured in cell supernatants by Western blotting and gelatin zymography. Results: Phagocytosis of HZ by human monocytes increased production of MIP-1 alpha, mRNA expression of MMP-9 and protein release of proMMP-9 and active MMP-9. All the HZ-enhancing effects on MMP-9 were abrogated by anti-human MIP-1alpha blocking antibodies and mimicked by recombinant human MIP-1alpha. Conclusions: The present work suggests a role for MIP-1alpha in the HZ-dependent enhancement of MMP-9 expression, release and activity observed in human monocytes, highlighting new detrimental effects of HZ-triggered proinflammatory response by phagocytic cells in falciparum malaria. [Copyright &y& Elsevier]

Abstract: Objective: To investigate the effect of malarial pigment (hemozoin, HZ) on expression of heat shock proteins (HSPs) and cell viability in human monocytes by using a stable cell line (THP-1 cells). Methods: THP-1 cells were fed with native HZ or treated with pro-apoptotic molecule gliotoxin for 9 h. Thereafter, the protein expression of HSP-27 and HSP-70 was evaluated by western blotting. Alternatively, HZ-fed cells were cultured up to 72 h and cell viability parameters (survival, apoptosis and necrosis rates) were measured by flow cytometric analysis. Results: HZ increased basal protein levels of HSP-27 without altering those of HSP-70 in THP-1 cells, and promoted long-term cell survival without inducing apoptosis. As expected, gliotoxin inhibited HSP-27 protein expression and promoted long-term cell apoptosis. Conclusions: Present data show that HZ prevents cell apoptosis and enhances the expression of anti-apoptotic HSP-27 in THP-1 cells, confirming the previous evidences obtained from HZ-fed immunopurified monocytes. Since the use of a stable cell line is pivotal to perform HSP-27 silencing experiments, monocytic THP-1 cells could be a good candidate line for such an approach, which is heavily required to clarify the role of HSP-27 in survival of impaired HZ-fed monocytes during falciparum malaria. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

An abstract of the article "Human lysozyme as a potential diagnostic marker in malaria: a mechanistic study of haemozoininduced monocyte degranulation," by Manuela Polimeni, Elena Valente, Elisabetta Aldieri, Amina Khadjavi, Giuliana Giribaldi, and Mauro Prato is presented.