Induction of Neuroinflammation and Neurotoxicity by Synthetic Hemozoin.

Hemozoin produced by Plasmodium falciparum during malaria infection has been linked to the neurological dysfunction in cerebral malaria. In this study, we determined whether a synthetic form of hemozoin (sHZ) produces neuroinflammation and neurotoxicity in cellular models. Incubation of BV-2 microglia with sHZ (200 and 400 µg/ml) induced significant elevation in the levels of TNFα, IL-6, IL-1ß, NO/iNOS, phospho-p65, accompanied by an increase in DNA binding of NF-κB. Treatment of BV-2 microglia with sHZ increased protein levels of NLRP3 with accompanying increase in caspase-1 activity. In the presence of NF-κB inhibitor BAY11-7082 (10 µM), there was attenuation of sHZ-induced release of pro-inflammatory cytokines, NO/iNOS. In addition, increase in caspase-1/NLRP3 inflammasome activation was blocked by BAY11-7082. Pre-treatment with BAY11-7082 also reduced both phosphorylation and DNA binding of the p65 sub-unit. The NLRP3 inhibitor CRID3 (100 µM) did not prevent sHZ-induced release of TNFα and IL-6. However, production of IL-1ß, NO/iNOS as well as caspase-1/NLRP3 activity was significantly reduced in the presence of CRID3. Incubation of differentiated neural progenitor (ReNcell VM) cells with sHZ resulted in a reduction in cell viability, accompanied by significant generation of cellular ROS and increased activity of caspase-6, while sHZ-induced neurotoxicity was prevented by N-acetylcysteine and Z-VEID-FMK. Taken together, this study shows that the synthetic form of hemozoin induces neuroinflammation through the activation of NF-κB and NLRP3 inflammasome. It is also proposed that sHZ induces ROS- and caspase-6-mediated neurotoxicity. These results have thrown more light on the actions of malarial hemozoin in the neurobiology of cerebral malaria.

Hemichorea caused by nonketotic hyperosmolar state: A case report and review of the literature.

Our report involves a case of hemichorea caused by the nonketotic hyperosmolar state. We have analyzed the clinical data and relevant features of a patient who presented herself to the Affiliated Hospital of Xuzhou Medical University. The patient had unilateral involuntary movements for 1 month. We discovered that her blood glucose levels were very high. The patient underwent computed tomography (CT), magnetic resonance imaging (MRI), and magnetic resonance angiography (MRA), indicating right basal ganglia lesion. Control of the patient's blood glucose plus supportive treatment resulted in a significant improvement of her clinical state.

African-centric TP53 variant increases iron accumulation and bacterial pathogenesis but improves response to malaria toxin.

A variant at amino acid 47 in human TP53 exists predominantly in individuals of African descent. P47S human and mouse cells show increased cancer risk due to defective ferroptosis. Here, we show that this ferroptotic defect causes iron accumulation in P47S macrophages. This high iron content alters macrophage cytokine profiles, leads to higher arginase level and activity, and decreased nitric oxide synthase activity. This leads to more productive intracellular bacterial infections but is protective against malarial toxin hemozoin. Proteomics of macrophages reveal decreased liver X receptor (LXR) activation, inflammation and antibacterial defense in P47S macrophages. Both iron chelators and LXR agonists improve the response of P47S mice to bacterial infection. African Americans with elevated saturated transferrin and serum ferritin show higher prevalence of the P47S variant (OR = 1.68 (95%CI 1.07-2.65) p = 0.023), suggestive of its role in iron accumulation in humans. This altered macrophage phenotype may confer an advantage in malaria-endemic sub-Saharan Africa.

Effects of heme proteins on nitric oxide levels and cell viability in isolated PMNs: a mechanism of toxicity.

Isolated human PMNs served as a model to determine oxyhemoglobin (oxyHb) binding and the effects of oxymyoglobin (oxyMb) or oxyHb on production of both nitric oxide (NO*) and superoxide (O2*-) and the resulting cytotoxicity. Physiologically relevant concentrations of NO* and H2O2 oxidized, to a similar extent, 2,7-dichlorodihydrofluorescein (DCFH) loaded into polymorphonuclear neutrophils (PMNs). Activation of PMNs with phorbol 12-myristate 13-acetate (PMA) markedly increased the internalization of extracellular oxyHb (10-250 microg/mL). OxyMb (10-300 microg/mL) or oxyHb (30-300 microg/mL) enhanced DCFH oxidation by a concentration-dependent mechanism in unstimulated, lipopolysaccharide (LPS) and tumor necrosis factor alpha (TNF-alpha)-, and PMA-stimulated PMNs. This increased DCFH oxidation was eliminated by NO* synthase inhibitors, glutathione and ascorbate, and was reduced by albumin. Nitrite accumulation in PMN filtrates mirrored NO*-induced DCF fluorescence. OxyMb-induced increases in NO* levels paralleled alterations in DNA and cell membrane damage and ATP levels in PMNs and co-cultured lymphocytes, and were attenuated by NO* synthase inhibitors. OxyMb eliminated extracellular O2*- at protein concentrations 100- to 1000-fold above those of superoxide dismutase. These results suggest that heme proteins bind and internalize into PMNs and increase NO*-induced damage in neighboring cells by inhibiting O2*(-)-scavenging of NO*. We propose a mechanism whereby heme protein-induced NO* levels may contribute to immunosuppression and increased infection rates associated with transfusions and cellular damage during inflammation.

On the pro-oxidant effects of haemozoin.

Haemozoin (Hz) is a haem aggregate produced in some blood-feeding organisms. There is a general belief that Hz formation would be a protective mechanism against haem toxicity. Here we show that when aggregated into Hz, haem is less deleterious than its free form. When haem was added to phosphatidylcholine (PC) liposomes, there was an intense stimulation of oxygen consumption, which did not occur when Hz was incubated with the same preparation. Evaluation of oxygen radical attack to lipids, by measurement of thiobarbituric acid reactive substances (TBARS), showed significantly lower levels of lipid peroxidation in samples containing PC liposomes incubated with Hz than with haem. However, TBARS production induced by Hz was much higher when using 2-deoxyribose (2-DR) as substrate, than with PC liposomes. Spin-trapping analysis by electron paramagnetic resonance (EPR) of Hz and tert-butylhydroperoxide (tert-BuOOH) showed that production of methoxyl and tert-butoxyl radicals was only slightly reduced compared to what was observed with haem. Interestingly, when large Hz crystals were used in 2-DR TBARS assays and tert-BuOOH EPR experiments, the pro-oxidant effects of Hz were strongly reduced. Moreover, increasing concentrations of Hz did not induce erythrocyte lysis, as occurred with haem. Thus, the reduced capacity of Hz to impose radical damage seems to result from steric hindrance of substrates to access the aggregated haem, that becomes less available to participate in redox reactions.

Increased levels of 4-hydroxynonenal in human monocytes fed with malarial pigment hemozoin. A possible clue for hemozoin toxicity.

In human monocytes, lipoperoxides were increased 3-fold at 2 h, 6-fold at 5 h and 7.5-fold at 12 h after hemozoin phagocytosis. 4-Hydroxynonenal (HNE) was also increased, reaching 40 nmol/10(10) cells at 2 h (approximate intracellular concentration [AIE] 8 microM) 230 nmol/10(10) cells at 5 h (AIE 46 microM) and 79 nmol/10(10) cells (AIE 16 microM) at 12 h. A moderate increase in HNE, approx. 20 nmol/10(10) cells (AIE 4 microM) was also observed after phagocytosis of anti-D IgG-opsonized erythrocytes. HNE in unfed controls was approx. 5 nmol/10(10) cells (AIE 1 microM) during the whole incubation period. An increased amount of protein kinase C (PKC)/HNE adduct was demonstrated in hemozoin-fed monocytes. Purified PKC was profoundly inhibited at HNE > 10 microM. The impairment of PKC previously observed in hemozoin-fed monocytes can thus be explained by direct interaction with increased HNE levels.

Mechanism of asbestos-mediated DNA damage: role of heme and heme proteins.

Several observations, including studies from this laboratory, demonstrate that asbestos generates free radicals in the biological system that may play a role in the manifestation of asbestos-related cytotoxicity and carcinogenicity. It has also been demonstrated that iron associated with asbestos plays an important role in the asbestos-mediated generation of reactive oxygen species. Exposure to asbestos leads to degradation of heme proteins such as cytochrome P450-releasing heme in cytosol. Our simulation experiments in the presence of heme show that such asbestos-released heme may increase lipid peroxidation and can cause DNA damage. Further, heme and horseradish peroxidase (HRP) can cause extensive DNA damage in the presence of asbestos and hydrogen peroxide/organic peroxide/hydroperoxides. HRP catalyzes oxidation reactions in a manner similar to that of prostaglandin H synthetase. Iron released from asbestos is only partially responsible for DNA damage. However, our studies indicate that DNA damage mediated by asbestos in vivo may be caused by a combination of effects such as the release and participation of iron, heme, and heme moiety of prostaglandin H synthetase in free radical generation from peroxides and hydroperoxides.

15(S)-hydroxyeicosatetraenoic acid (15-HETE), a product of arachidonic acid peroxidation, is an active component of hemozoin toxicity to monocytes.

Several studies have shown that human and murine hemozoin-fed phagocytes are functionally impaired. Unpurified hemozoin contains unspecifically attached unsaturated fatty acids such as arachidonic and linolenic acids. The presence in unpurified hemozoin of large quantities of ferric heme with small amounts of free iron makes hemozoin a generator of oxidative radicals capable of forming lipoperoxides or other breakdown products from polyunsaturated fatty acids. Here we show that delipidized hemozoin had reduced toxicity to monocytes. Phorbol myristate acetate (PMA)-elicited burst was poorly affected by delipidized hemozoin (ca. 17% and 21% burst inhibition by delipidized hemozoin vs ca. 75% and 65% burst inhibition by native hemozoin at 20 min or 17 h post-phagocytosis, respectively). Analysis of the lipid fraction isolated from native hemozoin by HPLC and chiral-phase HPLC showed equimolar amounts of 15(R)- and 15(S)-HETE (HETE, 15-hydroxy-6,8,11,13-eicosatetraenoic acid), most likely by-products of non-enzymatic peroxidation of arachidonic acid. The biologically active isomer, 15(S)-HETE, the product of 15-lipoxygenase, is a powerful mediator of inflammation and the effector of a large number of bioactions. 15(R,S)-HETE was found in native hemozoin (0.24 millimole/mole hemozoin heme), in supernatants of hemozoin-fed monocytes (87 nMol) and in hemozoin-fed monocytes (9.6 microMol). Approximately 84% of 15-HETE attached to hemozoin was in the esterified form. A large preponderance of esterified over free 15-HETE was also noted in supernatants of hemozoin-fed monocytes and in hemozoin-fed monocytes. In the latter cells, remarkable levels of the substance were attained. A dose-dependent curve of inhibition of PMA-elicited oxidative burst was observed. Assuming homogenuous distribution of 15-HETE in hemozoin-fed monocytes, 15(S)-HETE concentrations measured in hemozoin-fed monocytes (8 muMol) would bring about ca. 85% inhibition of PMA-elicited burst. In conclusion, derivatives of lipoperoxidation of unsaturated fatty acids such as 4-hydroxynonenal, 15-HETE and others now under study, appear to be relevant causes of hemozoin toxicity.

Hemozoin induces hepatic inflammation in mice and is differentially associated with liver pathology depending on the Plasmodium strain.

Malaria is a global disease that clinically affects more than two hundred million people annually. Despite the availability of effective antimalarials, mortality rates associated with severe complications are high. Hepatopathy is frequently observed in patients with severe malarial disease and its pathogenesis is poorly understood. Previously, we observed high amounts of hemozoin or malaria pigment in livers from infected mice. In this study, we investigated whether hemozoin is associated with liver injury in different mouse malaria models. C57BL/6J mice infected with the rodent parasites Plasmodium berghei ANKA, P. berghei NK65 or P. chabaudi AS had elevated serum liver enzymes without severe histological changes in the liver, in line with the observations in most patients. Furthermore, liver enzymes were significantly higher in serum of P. chabaudi AS-infected mice compared to mice infected with the P. berghei parasite strains and a strong positive correlation was found between hepatic hemozoin levels, hepatocyte damage and inflammation in the liver with P. chabaudi AS. The observed liver injury was only marginally influenced by the genetic background of the host, since similar serum liver enzyme levels were measured in infected C57BL/6J and BALB/c mice. Intravenous injection of P. falciparum-derived hemozoin in malaria-free C57BL/6J mice induced inflammatory gene transcription in the liver, suggesting that hemozoin may be involved in the pathogenesis of malaria hepatopathy by inducing inflammation.

Phagocytic uptake of oxidized heme polymer is highly cytotoxic to macrophages.

Apoptosis in macrophages is responsible for immune-depression and pathological effects during malaria. Phagocytosis of PRBC causes induction of apoptosis in macrophages through release of cytosolic factors from infected cells. Heme polymer or ß-hematin causes dose-dependent death of macrophages with LC50 of 132 µg/ml and 182 µg/ml respectively. The toxicity of hemin or heme polymer was amplified several folds in the presence of non-toxic concentration of methemoglobin. ß-hematin uptake in macrophage through phagocytosis is crucial for enhanced toxicological effects in the presence of methemoglobin. Higher accumulation of ß-hematin is observed in macrophages treated with ß-hematin along with methemoglobin. Light and scanning electron microscopic observations further confirm accumulation of ß-hematin with cellular toxicity. Toxicological potentiation of pro-oxidant molecules toward macrophages depends on generation of H2O2 and independent to release of free iron from pro-oxidant molecules. Methemoglobin oxidizes ß-hematin to form oxidized ß-hematin (ßH*) through single electron transfer mechanism. Pre-treatment of reaction mixture with spin-trap Phenyl-N-t-butyl-nitrone dose-dependently reverses the ß-hematin toxicity, indicates crucial role of ßH* generation with the toxicological potentiation. Acridine orange/ethidium bromide staining and DNA fragmentation analysis indicate that macrophage follows an oxidative stress dependent apoptotic pathway to cause death. In summary, current work highlights mutual co-operation between methemoglobin and different pro-oxidant molecules to enhance toxicity towards macrophages. Hence, methemoglobin peroxidase activity can be probed for subduing cellular toxicity of pro-oxidant molecules and it may in-turn make up for host immune response against the malaria parasite.

Structure and mechanism in salivary proteins from blood-feeding arthropods.

The saliva of blood-feeding arthropods contains rich mixtures of ligand binding proteins targeted at inhibiting hemostasis and inflammation in the host. Since blood feeding has evolved many times, different taxonomic groups utilize completely different families of proteins to perform similar tasks. Structural studies performed on a number of these proteins have revealed biologically novel and sophisticated mechanisms used to perform their functions. Here, the results of these structural and mechanistic studies are reviewed.

Suppression of erythropoiesis in malarial anemia is associated with hemozoin in vitro and in vivo.

Malarial anemia is a global public health problem and is characterized by a low reticulocyte response in the presence of life-threatening hemolysis. Although cytokines, in particular tumor necrosis factor-alpha (TNF-alpha), can suppress erythropoiesis, the grossly abnormal bone marrow morphology indicates that other factors may contribute to ineffective erythropoiesis. We hypothesized that the cytotoxic hemozoin (Hz) residues from digested hemoglobin (Hb) significantly contribute to abnormal erythropoiesis. Here, we show that not only isolated Hz, but also delipidated Hz, inhibits erythroid development in vitro in the absence of TNF-alpha. However, when added to cultures, TNF-alpha synergizes with Hz to inhibit erythropoiesis. Furthermore, we show that, in children with malarial anemia, the proportion of circulating monocytes containing Hz is associated with anemia (P < .001) and reticulocyte suppression (P = .009), and that this is independent of the level of circulating cytokines, including TNF-alpha. Plasma Hz is also associated with anemia (P < .001) and reticulocyte suppression (P = .02). Finally, histologic examination of the bone marrow of children who have died from malaria shows that pigmented erythroid and myeloid precursors are associated with the degree of abnormal erythroid development. Taken together, these observations provide compelling evidence for inhibition of erythropoiesis by Hz.

Destabilisation and subsequent lysis of human erythrocytes induced by Plasmodium falciparum haem products.

In falciparum malaria, both infected and uninfected red cells have structural and functional alterations. To investigate the mechanisms of these modifications, we studied the effects of two Plasmodium falciparum haem products (haematin and malaria pigment in the synthetic form beta-haematin) on isolated human red blood cells (RBCs) and purified RBC ghosts. A dose- and time-dependent incorporation of haematin into RBC ghosts and intact cells was observed, which was in proportion to the extent of haematin- induced haemolysis. RBCs pre-incubated with haematin were more sensitive to haemolysis induced by hypotonic shock, low pH, H2O2 or haematin itself. Haemolysis was not related to membrane lipid peroxidation and only partially to oxidation of protein sulphydryl groups and it could not be prevented by scavengers of lipid peroxidation or hydroperoxide groups. N-acetylcysteine partly protected the oxidation of SH groups and significantly reduced haemolysis. In contrast, beta-haematin was neither haemolytic nor oxidative towards protein sulphydryl groups. Beta-haematin did destabilise the RBC membrane, but to a lesser extent than haematin, inducing increased susceptibility to lysis caused by hypotonic medium, H2O2 or haematin. This study suggests that the destabilising effect of haematin and, to a much less extent, beta-haematin on the RBC membrane does not result from oxidative damage of membrane lipids but from direct binding or incorporation which may affect the reciprocal interactions between the membrane and cytoskeleton proteins. These changes could contribute to the reduced red cell deformability associated with severe malaria.

Intracellular targets in heme protein-induced renal injury.

We examined two potential intracellular targets in the glycerol model of acute renal failure, namely, the mitochondrion and the nucleus. Within three hours, alterations in mitochondrial function are already apparent. With either glutamate/malate or succinate/rotenone, state 3 and uncoupled respirations were decreased at three hours, and at 24 hours, such decrements were quite pronounced; in the presence of glutamate/malate, state 2 respiration was also depressed at 24 hours, while with succinate/rotenone state 2 was increased. Marked ultrastructural changes were observed in mitochondria studied at three hours, including the novel finding of degenerate mitochondria in autophagic vacuoles. Since the heme content in mitochondria was increased some tenfold within three hours, mitochondrial function was studied after exposure to concentrations of heme that reproduced such contents of heme: mitochondria initially displayed increased respiration, and subsequently, a persistent decline in oxygen consumption until oxygen consumption was virtually undetectable. With higher concentrations of heme, the early increase in oxygen consumption was blunted and the progressive decline in oxygen consumption was hastened. The antioxidant iron chelator, deferoxamine, prevented the early rise in oxygen consumption but did not prevent or delay the subsequent decline. We also assessed nuclear damage as a potential lesion in the glycerol model. DNA laddering was not observed at any time point. At 3 and 24 hours there was DNA injury by the TUNEL technique in the distal nephron but not in the proximal nephron. The 8-hydroxydeoxyguanosine/deoxyguanosine content was increased in the glycerol kidneys at 24 hours but not at three hours. At neither time point was evidence of apoptosis observed by light or electron microscopy. In studies undertaken in cell culture models, heme, at concentrations of 10 microM, failed to evince any such changes in LLC-PK1 cells, a cell line from the proximal tubule, or in MDCK cells, a cell line derived from the distal tubule. At concentrations of 50 microM, heme induced approximately 20% positivity in MDCK cells but none in LLC-PK1 cells by the TUNEL technique. We conclude that mitochondria and nuclei are prominent targets for injury in the glycerol model of acute renal failure. The presence of TUNEL-positive cells in the distal nephron but not at proximal sites in vivo underscores the increasing appreciation of the distinct responses of these nephron sites to nephrotoxic insults.

Malaria-parasitized erythrocytes and hemozoin nonenzymatically generate large amounts of hydroxy fatty acids that inhibit monocyte functions.

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.