Abstract
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 β-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. β-hematin uptake in macrophage through phagocytosis is crucial for enhanced toxicological effects in the presence of methemoglobin. Higher accumulation of β-hematin is observed in macrophages treated with β-hematin along with methemoglobin. Light and scanning electron microscopic observations further confirm accumulation of β-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 β-hematin to form oxidized β-hematin (βH*) through single electron transfer mechanism. Pre-treatment of reaction mixture with spin-trap Phenyl-N-t-butyl-nitrone dose-dependently reverses the β-hematin toxicity, indicates crucial role of βH* 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.
Highlights
Malaria caused by Plasmodium falciparum completes its life cycle in the vertebrate host human and invertebrate host mosquito [1,2]
We further explored whether Phenyl N-t-butylnitrone (PBN) is binding to the bH* and protecting macrophages from cytotoxic effects
Propogation of malaria parasite within host blood releases a mixture of different pro-oxidant molecules such as methemoglobin, heme, haemozoin and malaria toxins [5,6,7,30,47,48]
Summary
Malaria caused by Plasmodium falciparum completes its life cycle in the vertebrate host human and invertebrate host mosquito [1,2]. Rupture of parasite containing RBCs leads to release a large amount of hemoglobin/methemoglobin, free hemin, haemozoin, malaria toxins, and other uncharacterized metabolic by-products [5,6,7]. Macrophages exposed to haemozoin or b-hematin (synthetic haemozoin) is responsible for immune-depression during malaria It exhibits depression of phagocytosis, inhibition of phagosome/lysosome maturation and disturbance of pro-inflammatory/anti-inflammatory cytokine balance. Inflammation at brain site through aberrant activation of immune cells (T-cells or phagocytes) leads to leaky behavior of endothelial cells. It results in accumulation of parasitized RBCs and pathological complication of brain [26,27]. Pro-oxidant molecules released during malaria contribute in pathological complications of cerebral malaria, vascular complication and immune-depression following multiple mechanisms, but still it is not conclusive [28,29,30]
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