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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.

The lipid moiety of natural haemozoin (nHZ, malarial pigment) was previously shown to enhance expression and release of human monocyte matrix metalloproteinase-9 (MMP-9), and a major role for 15-(S,R)-hydroxy-6,8,11,13-eicosatetraenoic acid (15-HETE), a nHZ lipoperoxidation product, was proposed. Here, the underlying mechanisms were investigated, focusing on the involvement of mitogen-activated protein kinases (MAPKs). Results showed that nHZ promoted either early or late p38 MAPK phosphorylation; however, nHZ did not modify basal phosphorylation/expression ratios of extracellular signal-regulated kinase-1/2 and c-jun N-terminal kinase-1/2. 15-HETE mimicked nHZ effects on p38 MAPK, whereas lipid-free synthetic (s)HZ and delipidized (d)HZ did not. Consistently, both nHZ and 15-HETE also promoted phosphorylation of MAPK-activated protein kinase-2, a known p38 MAPK substrate; such an effect was abolished by SB203580, a synthetic p38 MAPK inhibitor. SB203580 also abrogated nHZ-dependent and 15-HETE-dependent enhancement of MMP-9 mRNA and protein (latent and activated forms) levels in cell lysates and supernatants. Collectively, these data suggest that in human monocytes, nHZ and 15-HETE upregulate MMP-9 expression and secretion through activation of p38 MAPK pathway. The present work provides new evidence on mechanisms underlying MMP-9 deregulation in malaria, which might be helpful to design new specific drugs for adjuvant therapy in complicated malaria., (Copyright © 2013 John Wiley & Sons, Ltd.)

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.

Natural hemozoin (nHZ), a lipid-bound ferriprotoporphyrin IX crystal produced by Plasmodium parasites after hemoglobin catabolism, seriously compromises the functions of human monocytes, and 15-hydroxyeicosatetraenoic acid (15-HETE) and 4-hydroxynonenal (4-HNE), two nHZ lipoperoxidation products, have been related to such a functional impairment. nHZ was recently shown to promote inflammation-mediated lysozyme release from human monocytes through p38 mitogen-activated protein kinase- (MAPK)- and nuclear factor (NF)-κB-dependent mechanisms. This study aimed at identifying the molecule of nHZ lipid moiety that was responsible for these effects. Results showed that 15-HETE mimicked nHZ effects on lysozyme release, whereas 4-HNE did not. 15-HETE-enhanced lysozyme release was abrogated by anti-TNF-α and anti-IL-1β-blocking antibodies and mimicked by recombinant cytokines; on the contrary, MIP-1α/CCL3 was not involved as a soluble mediator of 15-HETE effects. Moreover, 15-HETE early activated p38 MAPK and NF-κB pathways by inducing p38 MAPK phosphorylation; cytosolic I-κBα phosphorylation and degradation; NF-κB nuclear translocation and DNA-binding. Inhibition of both routes through chemical inhibitors (SB203580, quercetin, artemisinin, and parthenolide) prevented 15-HETE-dependent lysozyme release. Collectively, these data suggest that 15-HETE plays a major role in nHZ-enhanced monocyte degranulation., (Copyright © 2013 International Union of Biochemistry and Molecular Biology, Inc.)

Malarial pigment (natural haemozoin, HZ) is a ferriprotoporphyrin IX crystal produced by Plasmodium parasites after haemoglobin catabolism. HZ-fed human monocytes are functionally compromised, releasing increased amounts of pro-inflammatory molecules, including cytokines, chemokines and cytokine-related proteolytic enzyme Matrix Metalloproteinase-9 (MMP-9), whose role in complicated malaria has been recently suggested. In a previous work HZ was shown to induce through TNFalpha production the release of monocytic lysozyme, an enzyme stored in gelatinase granules with MMP-9. Here, the underlying mechanisms were investigated. Results showed that HZ lipid moiety promoted early but not late lysozyme release. HZ-dependent lysozyme induction was abrogated by anti-TNFalpha/IL-1 beta/MIP-1 alpha blocking antibodies and mimicked by recombinant cytokines. Moreover, HZ early activated either p38 MAPK or NF-kappaB pathways by inducing: p38 MAPK phosphorylation; cytosolic I-kappaB alpha phosphorylation and degradation; NF-kappaB nuclear translocation and DNA-binding. Inhibition of both routes through selected molecules (SB203580, quercetin, artemisinin, parthenolide) prevented HZ-dependent lysozyme release. These data suggest that HZ-triggered overproduction of TNFalpha, IL-1 beta and MIP-1 alpha mediates induction of lysozyme release from human monocytes through activation of p38 MAPK and NF-kappaB pathways, providing new evidence on mechanisms underlying the HZ-enhanced monocyte degranulation in falciparum malaria and the potential role for lysozyme as a new affordable marker in severe malaria.

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 © 2011 Hainan Medical College. Published by Elsevier B.V. All rights reserved.)

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-1β (IL-1β) 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], GROβ, GROγ, macrophage inflammatory protein 1α [MIP-1α], MIP-1β, 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-1β transcription, rapidly sustained by chemokine, TNF-α, MMP-9, and IL-1RA transcription and upregulation of HSP27 protein expression.

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.

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]