Macrophage Preconditioning with Synthetic Malaria Pigment Reduces Cytokine Production via Heme Iron-Dependent Oxidative Stress

Abstract

SUMMARY:Hemozoin (malaria pigment), a polymer of hematin (ferri-protoporphyrin IX) derived from hemoglobin ingested by intraerythrocytic plasmodia, modulates cytokine production by phagocytes. Mouse peritoneal macrophages (PM) fed with synthetic ␤-hematin (BH), structurally identical to native hemozoin, no longer produce tumor necrosis factor ␣ (TNF␣) and nitric oxide (NO) in response to lipopolysaccharide (LPS). Impairment of NO synthesis is due to inhibition of inducible nitric oxide synthase (iNOS) production. BH-mediated inhibition of PM functions cannot be ascribed to iron release from BH because neither prevention by iron chelators nor down-regulation of iron-regulatory protein activity was detected. Inhibition appears to be related to pigment-induced oxidative stress because (a) thiol compounds partially restored PM functions, (b) heme oxygenase (HO-1) and catalase mRNA levels were up-regulated, and (c) free radicals production increased in BH-treated cells. The antioxidant defenses of the cells determine the response to BH: microglia cells, which show a lower extent of induction of HO-1 and catalase mRNAs and lower accumulation of oxygen radicals, are less sensitive to the inhibitory effect of BH on cytokine production. Results indicate that BH is resistant to degradation by HO-1 and that heme-iron mediated oxidative stress may contribute to malaria-induced immunosuppression. This study may help correlate the different clinical manifestations of malaria, ranging from uncomplicated to severe disease, with dysregulation of phagocyte functions and promote better therapeutic strategies to counteract the effects of hemozoin accumulation. (Lab Invest 2000, 80:1781-1788.I ntraerythrocytic stage plasmodia digest hemoglobin in the food vacuole, where the globin is degraded and heme is detoxified, principally by polymerization/crystallization into hemozoin, an insoluble, microcrystalline derivative of ferri-protoporphyrin IX (␣ hematin, AH) (Bohle et al, 1997;Francis et al, 1997;Pagola et al, 2000;Slater et al, 1991). As parasites mature, hemozoin (malaria pigment) accumulates in the food vacuole and remains in the "residual body" after schizont rupture. Pigment is found inside host's phagocytes following ingestion of whole parasites or "residual bodies." Although a clinical correlation between the presence of pigment in circulating neutrophils and monocytes and disease severity is established (Nguyen et al, 1995), the nature of the interaction between pigment and phagocytes, as well as its contribution to the pathogenesis of malaria, and particularly its severe complications, are still controversial. Indeed, both pro-inflammatory (induction of tumor necrosis factor ␣ [TNF␣], interleukin-6 [IL-6], and chemotactic cytokines) (Pichyangkul et al, 1994;Prada et al, 1995;Sherry et al, 1995) and antiinflammatory activities (inhibition of respiratory burst and adhesion molecule expression and of TNF␣ and nitric oxide [NO] production) (Prada et al, 1995;Schwarzer et al, 1992;Taramelli et al, 1995Taramelli et al, , 1998 ha...

Hemozoin Increases IFN-γ-Inducible Macrophage Nitric Oxide Generation Through Extracellular Signal-Regulated Kinase- and NF-κB-Dependent Pathways

“…Therefore, it is possible that HZ leads to a localized negative or positive effect on M functions, depending on the cell susceptibility to ROS according to its tissue source. In fact, that HZ exerted a down-regulating effect on NO production by peritoneal M but not by microglial cells was attributed to differences in their antioxidant defenses (48). Additionally, because data from both human and rodent malaria indicate that M cytotoxic activities are maximal at the beginning of the infection and decline as the infection progresses (50), including the capacity to generate ROS (51), it is probable that HZ leads to M activation at the early stages of infection but after repetitive parasite cycles and subsequent M overloading with HZ, negative regulatory effects could be predominant.…”

“…Based on previous studies as well as on our data, a dual role for HZ on M modulation can be proposed. Acute phase malaria is characterized by high production of reactive oxygen species (ROS) (47), and HZ has been shown to increase ROS in M (48). Depending on the susceptibility to ROS, activation of the same signaling pathway has been found to lead to ROS-dependent transcription factor up-or down-regulation in different cell types (26,49).…”

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“…Though this suggests a higher phagocytic activity of the Kupffer cell-based RES of the liver, just the opposite may occur. Indeed, it has been shown in vitro that hemozoin accumulation in macrophages diminishes and eventually paralyses their activity (3,34,37,38,41,42). It appears rather likely that the testosterone-impaired closing of the liver is not able to efficiently restrain inundation with toxic parasite substances and inflammatory mediators, resulting in failure of the liver RES to act as an effector against P. chabaudi blood stages.…”

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