Regulation of Expression of Reactive Oxygen Intermediates During Plasmodium Infection to Reduce Immunopathology Provides a Possible Antioxidant Adjuvant to Enhance Anti-Malarial Drug Therapy

The life cycle of P. falciparum includes a symptomless, non-pathogenic liver stage of 7-10 days duration followed by the invasion of aged erythrocytes by infective forms, merozoites, which heralds the start of an intraerythrocytic vegetative growth cycle that is responsible for the pathogenic manifestations of disease (Figure 1). Erythrocytes rupture once mature schizonts are produced, which occurs 46-48 hours after invasion. Every erythrocyte releases between 16-32 merozoite progeny, each of which may bind to and enter a new erythrocyte to initiate a further cycle of asexual replication ⁴.

Figure 1.Schematic diagram showing the mechanisms of immunity against different life cycle stages of Plasmodium falciparum. * indicates those targeted immune responses in which reactive oxygen intermediates play an important role.

The human immune response against malaria infection is complex and depends on different stages of the life cycle ¹⁰ (Figure 1). Specific cytokines are released from human peripheral blood mononuclear cells which may activate the host’s monocytes, CD4⁺ and CD8⁺ T lymphocytes, natural killer (NK) cells and neutrophils to react to liver stage and blood stage parasites ¹¹. The immune mechanism to intraerythrocytic malaria parasites is not fully understood, yet a particular cytokine profile is known to be associated with protection that involves a pro-inflammatory response by CD4⁺ T cells of the helper (Th) 1 subset, with a prevalence of interferon (IFN)-γ, tumour necrosis factor (TNF)-α and interleukin (IL)-12 ^{12, 13, 14}. In contrast, the Th2 profile (notable for the production of IL-10, IL-5, IL-4 and transforming growth factor (TGF-β)) is noted to enhance severe conditions of the disease. However, since different immune responses act effectively at different stages of parasite elimination, there is no overall consensus on the respective pros and cons of these cytokine profiles ^{15, 16}.

Malaria infection activates the immune system resulting in the release of reactive oxygen intermediates (ROI) that induce oxidative damage which has the potential to kill parasite-infected hepatocytes and erythrocytes but also to cause cellular pathology ¹⁷. This triggers such severe effects on vital organs of the body as hepatomegaly, splenomegaly, endothelial and cognitive damage. ROI, together with reactive nitrogen intermediates (RNI) also linked with oxidative stress, are implicated in the development of systemic complications caused by malaria leading to the production of hydroxyl radicals in the liver that are thought to be a reason for the induction of oxidative stress and apoptosis ¹⁸. Additionally, erythrocytes infected with P. falciparum produce twice the concentration of hydroxyl radicals and hydrogen peroxide compared to uninfected erythrocytes ¹⁹. However, the body has various defence mechanisms to reduce the cellular effects of ROI including the production of antioxidants obtained either from the diet or synthesized via different intracellular mechanisms. Potential roles of an antioxidant are preventive or chain-breaking. Preventive antioxidants such as glutathione peroxidase, catalase, EDTA and vitamin C suppress the initial production of free radicals including ROI. So-called chain-breaking antioxidants like uric acid, superoxide dismutase and vitamin E inhibit damaging products of the ROI pathway ²⁰. Thus, antioxidants of both natural and synthetic origin possess the potential to confer host-protective adjuvant effects on antimalarial therapy that may result in reduced immunopathology during malaria infection.

The mechanisms of action of ROI and RNI involve reduction or elimination of parasite load (Figure 1), which is the process exploited by most anti-malaria drugs for their activity. These reactive intermediates have a great influence on immune responses by activating or inhibiting the action of certain transcription factors or cytokines and also by modifying pathways of programmed cell death ²¹. An elevated level of nitric oxide (NO) results in immunosuppression and is causally linked to the development of cerebral malaria ²². However, as free radicals NO metabolites contribute to the destruction of Plasmodium^{23, 24}. Malarial pigment is one of the few parasite-derived molecules that are known to elicit production of NO. Haemozoin is a potent inducer of NO generation in macrophages via the action of nuclear factor (NF)-κB and extracellular signal regulated by kinase (ERK). Haemozoin is associated with the mediator of IFN-γ mRNA synthesis through the enzyme inducible nitric oxide synthase (iNOS). During the hepatic stage of infection, defence mechanisms are linked to the release of IFN-γ by NK cells and NO synthesis ²⁵. Haemozoin not only induces NO but is also responsible for macrophage activation by processes that are partially dependent on NO ²⁶ and on other ROI/RNI such as the superoxide radical and hydrogen peroxide ²⁷.

On a related theme, an elevated concentration of iNOS in human monocytes has not been linked with an exacerbation of malaria. Studies conducted on spleen cells from P. berghei-infected mice either resistant or susceptible to cerebral malaria suggested that the expression of cytokines and NO increases in cells of resistant animals compared to their susceptible counterparts ²⁸. In addition, expression of a marker of TNF-α activity was also raised in resistant mice, suggesting that activation of macrophages is much greater in resistant animals ²⁹. These findings corroborate radiometric assays used to quantify the anti-plasmodial effect of phagocytes. These prior studies showed that TNF-α increases the release of ROI by neutrophils infected by P. falciparum, which are toxic and contribute to the elimination of the parasite ³⁰.

Derived from host erythrocytes, haemoglobin is a potential source of free radical synthesis in malaria. The parasite’s utilization of this protein molecule as a source of amino acids to meet its nutritional requirements during the erythrocytic stage of its life cycle results in liberation of extensive amounts of haem into the circulation ³¹. However these haem groups induce intravascular oxidative stress due to the presence of Fe²⁺-associated groups which cause metabolic flux in erythrocytes and endothelial cells and facilitate the internalization of the parasite in tissues such as the brain and liver ³². Moreover, free haem can activate neutrophil migration and ROI/RNI production with the help of the inhibitory Gα protein-coupled receptor that further activates protein kinase C, thus enhancing the inflammatory response ³³ and delaying apoptosis, thereby contributing to the immunosuppression induced by malaria infection ³⁴. Recently, it was shown that a transient oxidative insult to wild-type erythrocytes prior to infection with P. falciparum promotes phenotypic characters associated with the protective traits of haemoglobinopathic and fetal erythrocytes ³⁵. This implies abnormal host actin remodelling and reduced cytoadherence are steps in a malaria host-protective pathway initiated by the redox imbalance that is inherent to sickle cell trait and other common hereditary blood disorders.

Of various cytokines examined, an increase in granulocyte-macrophage colony-stimulating factor (GM-CSF) has been correlated with a reduction of parasitaemia and with oxidative stress ¹³. GM-CSF is a cytokine that is known to have stimulatory action on granulocytes and macrophages and that helps to enhance the number and activity of both of these cells, working effectively to provide cellular immunity against blood stage malaria. Several studies have reported that administration of GM-CSF, individually or in combination with other factors, protects experimental models from infection. Similarly, mice lacking the ability to synthesize GM-CSF have an impaired anti-Plasmodium immune response. Such research suggests a possible relationship between GM-CSF and oxidative stress. IL-4 and GM-CSF receptors may be altered by lipid peroxidation products derived from haemozoin that are released upon rupture of parasitized erythrocytes. These include, for example, 4-hydroxynonenal (4-HNE). IL-4 and GM-CSF are activators of the differentiation of haemozoin-loaded monocytes into dendritic cells which can be inhibited by 4-HNE and is an important immunosuppressive mechanism in malaria ³⁶. In immature dendritic cells this appears to be linked to expression of peroxisome proliferator-activated receptor gamma (PPAR-γ) by cells loaded with haemozoin after administration of ¹⁵ S-hydroxyeicosatetraenoic acid – which is a PPAR-γ ligand produced by haemozoin via peroxidation caused by haem – that prevents the differentiation of these cells ³⁷. A relationship exists between GM-CSF and NO, illustrated by pre-treatment with GM-CSF and methionine encephalin (TGG) helping to protect mice from malaria. Conversely, for mice pre-treated with iNOS inhibitors the mortality rate is enhanced significantly, showing that the protection exerted by GM-CSF/TGG is at least partially due to NO.

Both host and parasites are placed under oxidative stress during a malaria infection. ROI are multifunctional molecules associated with host defence, hormone biosynthesis, mitogenesis, necrosis, apoptosis and gene expression ³⁸. Increased concentrations of ROI are produced during degradation of haemoglobin within the parasite and by activated neutrophils in the peripheral blood of the host. In cases of severe malaria, ROI are produced by parasite haemoglobin metabolism in erythrocytes, nicotinamide adenine dinucleotide phosphate oxidase in phagocytes, and iNOS in response to deficiency of arginine or cofactor tetrahydrobiopterin substrate. Plasma haemoglobin produced upon erythrocyte lysis can also catalyze the generation of ROI. Patients suffering from severe malaria have enhanced level of ROI products in their urine ³⁹, reduced deformability of erythrocytes under shear stress and decreased α-tocopherol in erythrocyte membranes ⁴⁰. ROI may bestow both both protective and pathological effects on the host during malaria. After administration of pro-oxidants, enhanced ROI production is directed against intra-erythrocytic parasites. The utility of ROI to the innate immune response was first investigated in phagocytic cells by examining ROI production by NADPH oxidases (NOX) followed by pathogen killing ⁴¹. During infection erythrocytes are exposed to continuous oxidative stress. Univalent reduction of oxygen occurs, resulting in generation of a series of highly reactive cytotoxic oxygen species such as superoxide, hydrogen peroxide and hydroxyl. This causes a wide spectrum of cell damage including inactivation of enzymes, lipid peroxidation, damage to DNA and modification of intracellular oxidation-reduction states ¹³.

Many protective mechanisms have evolved to reduce the harmful effects of free radicals on cells. These include antioxidants like glutathione (GSH) and also enzymes like glutathione-S-transferase (GST), superoxide dismutase (SOD) and catalase that catalyse the dismutation of hydrogen peroxideand superoxide anions to water at the expense of GSH ⁴². Reduced GSH is a tripeptide with an intracellular non-protein free sulfhydryl group, and is essential to overcome oxidative stress and thus to maintain the normal reduced state inside the cell. NADPH generated by glucose 6-phosphate dehydrogenase in the pentose phosphate pathway reduces oxidized glutathione. Cells with reduced levels of glucose 6-phosphate dehydrogenase are very prone to oxidative stress. Erythrocytes experience very acute stress because, without mitochondria, there is no other means of generating reducing power.

Human erythrocytes are rich in membrane sulfhydryl groups which play a major role in maintenance of oxidation-reduction status of the cell. A greater concentration of sulfhydryl groups in oxidatively stressed erythrocytes occurs because of their reduced GST activity ⁴³. A longer association of toxic agents with sulfhydryl groups due to the decrease in GST activity may lead to their intraerythrocytic accumulation ⁴⁴. Reduced levels of SOD and catalase in blood samples taken from malaria-infected patients resulted in heightened susceptibility of the erythrocytes to cell damage.

The current anti-malarial drugs of choice often have a harmful side-effect, such as reported in memory impairment after cerebral malaria. As a consequence, recent studies have aimed to use antioxidants, alone or in combination with anti-malarials, as a potential therapeutic method focused at reducing Plasmodium-induced oxidative stress and its associated complications ⁴⁵. However, some anti-malarials act by triggering oxidative stress; hence, the practical application of this strategy often yields conflicting outcomes. In general, researched antioxidants are. The effects on Plasmodium parasites in vitro of the antioxidants desferroxamine, vitamins C and E, folate and N-acetylcystein have been studied by direct administration and as adjunct therapy alongside standard anti-malarial regimens. The therapeutic relevance of antioxidants in malaria treatment depends on the targeted aspect of malaria pathology ⁴⁶.