Gordon A. Awandare

Differential regulation of beta-chemokines in children with Plasmodium falciparum malaria.

ABSTRACT Chemokines regulate the host immune response to a variety of infectious pathogens. Since the role of chemokines in regulating host immunity in children with Plasmodium falciparum malaria has not previously been reported, circulating levels of β-chemokines (MIP-1α, MIP-1β, and RANTES) and their respective transcriptional profiles in ex vivo peripheral blood mononuclear cells (PBMCs) were investigated. Peripheral blood MIP-1α and MIP-1β levels were significantly elevated in mild and severe malaria, while RANTES levels decreased with increasing disease severity. β-Chemokine gene expression profiles in blood mononuclear cells closely matched those of circulating β-chemokines, illustrating that PBMCs are a primary source for the observed pattern of β-chemokine production during acute malaria. Statistical modeling revealed that none of the chemokines was significantly associated with either parasitemia or anemia. Additional investigations in healthy children with a known history of malaria showed that children with prior severe malaria had significantly lower baseline RANTES production than children with a history of mild malaria, suggesting inherent differences in the ability to produce RANTES in these two groups. Baseline MIP-1α and MIP-1β did not significantly differ between children with prior severe malaria and those with mild malaria. Additional in vitro experiments in PBMCs from healthy, malaria-naïve donors revealed that P. falciparum-derived hemozoin (Hz; malarial pigment) and synthetic Hz (β-hematin) promote a similar pattern of β-chemokine gene expression. Taken together, the results presented here demonstrate that children with severe malaria have a distinct profile of β-chemokines characterized by increased circulating levels of MIP-1α and MIP-1β and decreased RANTES. Altered patterns of circulating β-chemokines result, at least in part, from Hz-induced changes in β-chemokine gene expression in blood mononuclear cells.

Genomic epidemiology of artemisinin resistant malaria.

The current epidemic of artemisinin resistant Plasmodium falciparum in Southeast Asia is the result of a soft selective sweep involving at least 20 independent kelch13 mutations. In a large global survey, we find that kelch13 mutations which cause resistance in Southeast Asia are present at low frequency in Africa. We show that African kelch13 mutations have originated locally, and that kelch13 shows a normal variation pattern relative to other genes in Africa, whereas in Southeast Asia there is a great excess of non-synonymous mutations, many of which cause radical amino-acid changes. Thus, kelch13 is not currently undergoing strong selection in Africa, despite a deep reservoir of variations that could potentially allow resistance to emerge rapidly. The practical implications are that public health surveillance for artemisinin resistance should not rely on kelch13 data alone, and interventions to prevent resistance must account for local evolutionary conditions, shown by genomic epidemiology to differ greatly between geographical regions.

Complement Receptor 1 Is a Sialic Acid-Independent Erythrocyte Receptor of Plasmodium falciparum

Plasmodium falciparum is a highly lethal malaria parasite of humans. A major portion of its life cycle is dedicated to invading and multiplying inside erythrocytes. The molecular mechanisms of erythrocyte invasion are incompletely understood. P. falciparum depends heavily on sialic acid present on glycophorins to invade erythrocytes. However, a significant proportion of laboratory and field isolates are also able to invade erythrocytes in a sialic acid-independent manner. The identity of the erythrocyte sialic acid-independent receptor has been a mystery for decades. We report here that the complement receptor 1 (CR1) is a sialic acid-independent receptor for the invasion of erythrocytes by P. falciparum. We show that soluble CR1 (sCR1) as well as polyclonal and monoclonal antibodies against CR1 inhibit sialic acid-independent invasion in a variety of laboratory strains and wild isolates, and that merozoites interact directly with CR1 on the erythrocyte surface and with sCR1-coated microspheres. Also, the invasion of neuraminidase-treated erythrocytes correlates with the level of CR1 expression. Finally, both sialic acid-independent and dependent strains invade CR1 transgenic mouse erythrocytes preferentially over wild-type erythrocytes but invasion by the latter is more sensitive to neuraminidase. These results suggest that both sialic acid-dependent and independent strains interact with CR1 in the normal red cell during the invasion process. However, only sialic acid-independent strains can do so without the presence of glycophorin sialic acid. Our results close a longstanding and important gap in the understanding of the mechanism of erythrocyte invasion by P. falciparum that will eventually make possible the development of an effective blood stage vaccine.

Role of monocyte-acquired hemozoin in suppression of macrophage migration inhibitory factor in children with severe malarial anemia.

ABSTRACT Severe malarial anemia (SMA), caused by Plasmodium falciparum infections, is one of the leading causes of childhood mortality in sub-Saharan Africa. Although the molecular determinants of SMA are largely undefined, dysregulation in host-derived inflammatory mediators influences disease severity. Macrophage migration inhibitory factor (MIF) is an important regulator of innate inflammatory responses that has recently been shown to suppress erythropoiesis and promote pathogenesis of SMA in murine models. To examine the role of MIF in the development of childhood SMA, peripheral blood MIF production was examined in Kenyan children (aged <3 years, n = 357) with P. falciparum malarial anemia. All children in the study were free from bacteremia and human immunodeficiency virus type 1. Since deposition of malarial pigment (hemozoin [Hz]) contributes to suppression of erythropoiesis, the relationship between MIF concentrations and monocytic acquisition of Hz was also examined in vivo and in vitro. Circulating MIF concentrations declined with increasing severity of anemia and significantly correlated with peripheral blood leukocyte MIF transcripts. However, MIF concentrations in peripheral blood were not significantly associated with reticulocyte production. Multivariate regression analyses, controlling for age, gender, and parasitemia, further revealed that elevated levels of pigment-containing monocytes (PCM) was associated with SMA and decreased MIF production. In addition, PCM levels were a better predictor of hemoglobin and MIF concentrations than parasite density. Additional experiments in malaria-naive individuals demonstrated that hemozoin caused both increased and decreased MIF production in cultured peripheral blood mononuclear cells (PBMC) in a donor-specific manner, independent of apoptosis. However, PBMC MIF production in children with acute malaria progressively declined with increasing anemia severity. Results presented here demonstrate that acquisition of hemozoin by monocytes is associated with suppression of peripheral blood MIF production and enhanced severity of anemia in childhood malaria.

Increased Levels of Inflammatory Mediators in Children with Severe Plasmodium falciparum Malaria with Respiratory Distress

BACKGROUND Respiratory distress (RD), a symptom of underlying metabolic acidosis, has been identified as a major risk factor for mortality in children with severe malaria in Africa, yet the molecular mediators involved in the pathogenesis of RD have not been identified. METHODS We studied circulating levels of mediators of inflammation--including the cytokines tumor necrosis factor (TNF)- alpha and interleukin (IL)-10; the chemokines macrophage inflammatory protein (MIP)-1 alpha , MIP-1 beta , and IL-8; and the immune activation marker neopterin--in children with RD, severe malarial anemia (SMA), cerebral malaria (CM), and uncomplicated malaria (UM). RESULTS Children with RD had significantly higher plasma levels of TNF- alpha , IL-10, and neopterin and a significantly higher TNF- alpha : IL-10 ratio than those without RD. In addition, the results demonstrated that, relative to UM, CM was associated with increased levels of TNF- alpha and decreased levels of MIP-1 alpha , whereas SMA was associated with decreased levels of IL-10. Circulating levels of neopterin were inversely correlated with hemoglobin, whereas levels of MIP-1 beta were positively correlated with parasitemia. CONCLUSIONS We conclude that distinct clinical presentations of severe malaria are associated with specific patterns of inflammatory mediators. In particular, we show, to our knowledge for the first time, that patients with malaria and RD have a strong and unbalanced proinflammatory response that may be involved in the pathogenesis of the underlying metabolic acidosis.

MIF (Macrophage Migration Inhibitory Factor) Promoter Polymorphisms and Susceptibility to Severe Malarial Anemia

BACKGROUND Severe malarial anemia (SMA) resulting from Plasmodium falciparum infection is one of the leading causes of childhood mortality in sub-Saharan Africa. The innate immune mediator macrophage migration inhibitory factor (MIF) plays a critical role in the pathogenesis of SMA. METHODS To investigate the influence of MIF genetic variation on susceptibility to SMA, haplotypes of the MIF -173G/C and -794CATT5-8 polymorphisms were examined in a cohort of Kenyan children. RESULTS A statistically significant relationship between increasing frequencies of longer CATT repeats at -794 and increasing severity of malarial anemia was observed. In addition, there was a strong association between lower MIF concentrations and longer CATT repeats. Multivariate logistic regression analyses demonstrated that the 6G haplotype (ie, MIF -794CATT6/-173G) was associated with protection against SMA, whereas carriers of the 7C or 8C haplotype had increased risk of developing SMA. Furthermore, carriers of the 7C or 8C haplotype had reduced plasma MIF levels during acute disease. CONCLUSIONS The findings demonstrate that variation in the MIF promoter influences susceptibility to SMA and peripheral MIF production. However, the MIF -173 and -794 polymorphisms appear to have both independent and interactive effects on different measures of disease severity, suggesting that MIF plays a complex role in malarial pathogenesis.

A macrophage migration inhibitory factor promoter polymorphism is associated with high-density parasitemia in children with malaria.

Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that regulates innate and adaptive immune responses to bacterial and parasitic infections. Functional promoter variants in the MIF gene influence susceptibility to inflammatory diseases in Caucasians. As the role of genetic variation in the MIF gene in conditioning malaria disease outcomes is largely unexplored, the relationship between a G to C transition at MIF −173 and susceptibility to high-density parasitemia (HDP) and severe malarial anemia (SMA) was examined in Kenyan children (aged 3–36 months; n=477) in a holoendemic Plasmodium falciparum transmission region. In a multivariate model, controlling for age, gender, HIV-1 status, and sickle-cell trait, MIF −173CC was associated with an increased risk of HDP compared to MIF −173GG. No significant associations were found between MIF −173 genotypic variants and susceptibility to SMA. Additional studies demonstrated that homozygous G alleles were associated with lower basal circulating MIF levels relative to the GC group. However, stimulation of cultured peripheral blood mononuclear cells with malarial pigment (hemozoin) increased MIF production in the GG group and decreased MIF production in the GC group. Thus, variability at MIF −173 is associated with functional changes in MIF production and susceptibility to HDP in children with malaria.

Mechanisms of erythropoiesis inhibition by malarial pigment and malaria-induced proinflammatory mediators in an in vitro model

One of the commonest complications of Plasmodium falciparum malaria is the development of severe malarial anemia (SMA), which is, at least in part, due to malaria‐induced suppression of erythropoiesis. Factors associated with suppression of erythropoiesis and development of SMA include accumulation of malarial pigment (hemozoin, PfHz) in bone marrow and altered production of inflammatory mediators, such as tumor necrosis factor (TNF)‐α, and nitric oxide (NO). However, studies investigating the specific mechanisms responsible for inhibition of red blood cell development have been hampered by difficulties in obtaining bone marrow aspirates from infants and young children, and the lack of reliable models for examining erythroid development. As such, an in vitro model of erythropoiesis was developed using CD34+ stem cells derived from peripheral blood to examine the effects of PfHz, PfHz‐stimulated peripheral blood mononuclear cell (PBMC)‐conditioned media (CM‐PfHz), TNF‐α, and NO on erythroid cell development. PfHz only slightly suppressed erythroid cell proliferation and maturation marked by decreased expression of glycophorin A (GPA). On the other hand, CM‐PfHz, TNF‐α, and NO significantly inhibited erythroid cell proliferation. Furthermore, decreased proliferation in cells treated with CM‐PfHz and NO was accompanied by increased apoptosis of erythropoietin‐stimulated CD34+ cells. In addition, NO significantly inhibited erythroid cell maturation, whereas TNF‐α did not appear to be detrimental to maturation. Collectively, our results demonstrate that PfHz suppresses erythropoiesis by acting both directly on erythroid cells, and indirectly via inflammatory mediators produced from PfHz‐stimulated PBMC, including TNF‐α and NO.Am. J. Hematol. 86:155–162, 2011.

Naturally acquired hemozoin by monocytes promotes suppression of RANTES in children with malarial anemia through an IL-10-dependent mechanism.

Regulated upon activation, normal T-cell expressed, and secreted (RANTES, CCL-5) is an important immunoregulatory mediator that is suppressed in children with malarial anemia (MA). Although pro-inflammatory (e.g., TNF-alpha, IL-1beta and IFN-gamma) and anti-inflammatory (e.g., IL-4, IL-10 and IL-13) cytokines regulate RANTES production, their effect on RANTES in children with MA has not been determined. Since intraleukocytic malarial pigment, hemozoin (Hz), causes dysregulation in chemokine and cytokine production, the impact of naturally acquired Hz (pfHz) on RANTES and RANTES-regulatory cytokines (TNF-alpha, IFN-gamma, IL-1beta, IL-4, IL-10, and IL-13) was examined. Circulating RANTES levels progressively declined with increasing levels of pigment-containing monocytes (PCM) (P=0.035). Additional experiments in cultured peripheral blood mononuclear cells (PBMC) showed that monocytic acquisition of pfHz (in vivo) was associated with suppression of RANTES under baseline (P=0.001) and stimulated conditions (P=0.072). Although high PCM levels were associated with decreased circulating IFN-gamma (P=0.003) and IL-10 (P=0.010), multivariate modeling revealed that only PCM (P=0.048, beta=-0.171) and IL-10 (P<0.0001, beta=-0.476) were independently associated with RANTES production. Subsequent in vitro experiments revealed that blockade of endogenous IL-10 significantly increased RANTES production (P=0.028) in PBMC from children with naturally acquired Hz. Results here demonstrate that monocytic acquisition of Hz suppresses RANTES production in children with MA through an IL-10-dependent mechanism.

Polymorphic Variability in the Interleukin (IL)-1β Promoter Conditions Susceptibility to Severe Malarial Anemia and Functional Changes in IL-1β Production

Interleukin (IL)-1beta is a cytokine released as part of the innate immune response to Plasmodium falciparum. Because the role played by IL-1beta polymorphic variability in conditioning the immunopathogenesis of severe malarial anemia (SMA) remains undefined, relationships between IL-1beta promoter variants (-31C/T and -511A/G), SMA (hemoglobin [Hb] level <6.0 g/dL), and circulating IL-1beta levels were investigated in children with parasitemia (n= 566) from western Kenya. The IL-1beta promoter haplotype -31C/-511A (CA) was associated with increased risk of SMA (Hb level <6.0 g/dL; odds ratio [OR], 1.98 [95% confidence interval {CI}, 1.55-2.27]; P < .05) and reduced circulating IL-1beta levels (p <.05). The TA (-31T/-511A) haplotype was nonsignificantly associated with protection against SMA (OR, 0.52 [95% CI, 0.18-1.16]; p =.11) and elevated IL-1beta production ( p<.05). Compared with the non-SMA group, children with SMA had significantly lower IL-1beta levels and nonsignificant elevations in both IL-1 receptor antagonist (IL-1Ra) and the ratio of IL-1Ra to IL-1beta. The results presented demonstrate that variation in IL-1beta promoter conditions susceptibility to SMA and functional changes in circulating IL-1beta levels.