Reduced Parasite Burden in Children with Falciparum Malaria and Bacteremia Coinfections: Role of Mediators of Inflammation.

Bacteremia and malaria coinfection is a common and life-threatening condition in children residing in sub-Saharan Africa. We previously showed that coinfection with Gram negative (G[-]) enteric Bacilli and Plasmodium falciparum (Pf[+]) was associated with reduced high-density parasitemia (HDP, >10,000 parasites/µL), enhanced respiratory distress, and severe anemia. Since inflammatory mediators are largely unexplored in such coinfections, circulating cytokines were determined in four groups of children (n = 206, aged <3 yrs): healthy; Pf[+] alone; G[-] coinfected; and G[+] coinfected. Staphylococcus aureus and non-Typhi Salmonella were the most frequently isolated G[+] and G[-] organisms, respectively. Coinfected children, particularly those with G[-] pathogens, had lower parasite burden (peripheral and geometric mean parasitemia and HDP). In addition, both coinfected groups had increased IL-4, IL-5, IL-7, IL-12, IL-15, IL-17, IFN-γ, and IFN-α and decreased TNF-α relative to malaria alone. Children with G[-] coinfection had higher IL-1ß and IL-1Ra and lower IL-10 than the Pf[+] group and higher IFN-γ than the G[+] group. To determine how the immune response to malaria regulates parasitemia, cytokine production was investigated with a multiple mediation model. Cytokines with the greatest mediational impact on parasitemia were IL-4, IL-10, IL-12, and IFN-γ. Results here suggest that enhanced immune activation, especially in G[-] coinfected children, acts to reduce malaria parasite burden.

Suppressed circulating bicyclo-PGE2 levels and leukocyte COX-2 transcripts in children co-infected with P. falciparum malaria and HIV-1 or bacteremia.

In holoendemic Plasmodium falciparum transmission regions, malarial anemia is a leading cause of childhood morbidity and mortality. Identifying biomarkers of malaria disease severity is important for identifying at-risk groups and for improved understanding of the molecular pathways that influence clinical outcomes. We have previously shown that decreased cyclooxygenase (COX)-2-derived prostaglandin E2 (PGE2) levels are associated with enhanced clinical severity in cerebral malaria, malarial anemia, and malaria during pregnancy. Since children with malaria often have increased incidence of additional infections, such as bacteremia and HIV-1, we extend our previous findings by investigating COX-2 and PGE2 in children with falciparum malaria and co-infection with either bacteremia or HIV-1. Plasma bicyclo-PGE2/creatinine levels and peripheral blood COX-2 transcripts were significantly reduced in co-infected children relative to those with malaria mono-infection. Furthermore, suppression of circulating bicyclo-PGE2 was significantly associated with reduced hemoglobin levels in both mono- and co-infected children with malaria, suggesting that bicyclo-PGE2 may represent both a marker and mediator of malaria pathogenesis.

Reduced systemic bicyclo-prostaglandin-E2 and cyclooxygenase-2 gene expression are associated with inefficient erythropoiesis and enhanced uptake of monocytic hemozoin in children with severe malarial anemia.

In holoendemic Plasmodium falciparum transmission areas, severe malaria primarily occurs in children aged <48 months and manifests as severe malarial anemia [SMA; hemoglobin (Hb) < 6.0 g/dL]. Induction of high levels of prostaglandin-E(2) (PGE(2)) through inducible cyclooxygenase-2 (COX-2) is an important host-defense mechanism against invading pathogens. We have previously shown that COX-2-derived PGE(2) levels are reduced in children residing in hyperendemic transmission regions with cerebral malaria and in those with mixed sequelae of anemia and hyperparasitemia. Our in vitro studies further demonstrated that reduced PGE(2) was due to downregulation of COX-2 gene products following phagocytosis of malarial pigment (hemozoin, PfHz). However, as COX-2-PGE(2) pathways and the impact of naturally acquired PfHz on erythropoietic responses have not been determined in children with SMA, plasma and urinary bicyclo-PGE(2)/creatinine and leukocytic COX-2 transcripts were determined in parasitized children (<36 months) stratified into SMA (n = 36) and non-SMA (Hb ≥ 6.0 g/dL; n = 38). Children with SMA had significantly reduced plasma (P = 0.001) and urinary (P < 0.001) bicyclo-PGE(2)/creatinine and COX-2 transcripts (P = 0.007). There was a significant positive association between Hb and both plasma (r = 0.363, P = 0.002) and urinary (r = 0.500, P = 0.001)] bicyclo-PGE(2)/creatinine. Furthermore, decreased systemic bicyclo-PGE(2)/creatinine was associated with inefficient erythropoiesis (i.e., reticulocyte production index; RPI < 2.0, P = 0.026). Additional analyses demonstrated that plasma (P = 0.031) and urinary (P = 0.070) bicyclo-PGE(2)/creatinine and COX-2 transcripts (P = 0.026) progressively declined with increasing concentrations of naturally acquired PfHz by monocytes. Results presented here support a model in which reduced COX-2-derived PGE(2), driven in part by naturally acquired PfHz by monocytes, promotes decreased erythropoietic responses in children with SMA.

Reduced interferon (IFN)-α conditioned by IFNA2 (-173) and IFNA8 (-884) haplotypes is associated with enhanced susceptibility to severe malarial anemia and longitudinal all-cause mortality.

Severe malarial anemia (SMA) is a leading cause of pediatric morbidity and mortality in holoendemic Plasmodium falciparum transmission areas. Although dysregulation in cytokine production is an important etiology of SMA, the role of IFN-α in SMA has not been reported. As such, we investigated the relationship between IFN-α promoter polymorphisms [i.e., IFNA2 (A-173T) and IFNA8 (T-884A)], SMA, and functional changes in IFN-α production in children (n = 663; <36 months) residing in a holoendemic P. falciparum transmission region of Kenya. Children with SMA had lower circulating IFN-α than malaria-infected children without severe anemia (P = 0.025). Multivariate logistic regression analyses revealed that heterozygosity at -884 (TA) was associated with an increased risk of SMA [OR 2.80 (95 % CI 1.22-6.43); P = 0.015] and reduced IFN-α relative to wild type (TT; P = 0.038). Additional analyses demonstrated that carriage of the -173T/-884A (TA) haplotype was associated with increased susceptibility to SMA [OR 3.98 (95 % CI 1.17-13.52); P = 0.026] and lower IFN-α (P = 0.031). Follow-up of these children for 36 months revealed that carriers of TA haplotype had greater all-cause mortality than non-carriers (P < 0.001). Generation of reporter constructs showed that the IFNA8 wild-type -884TT exhibited higher levels of luciferase expression than the variant alleles (P < 0.001). Analyses of malaria-associated inflammatory mediators demonstrated that carriers of TA haplotype had altered production of IL-1ß, MIG, and IL-13 compared to non-carriers (P < 0.050). Thus, variation at IFNA2 -173 and IFNA8 -884 conditions reduced IFN-α production, and increased susceptibility to SMA and mortality.

Relationship between inflammatory mediator patterns and anemia in HIV-1 positive and exposed children with Plasmodium falciparum malaria.

Anemia is the primary hematological manifestation of both Plasmodium falciparum malaria and HIV-1 in pediatric populations in sub-Saharan Africa. We have previously shown that HIV-1 positive and exposed children have greater risk of developing severe anemia (hemoglobin, Hb <6.0 g dL⁻¹) during acute malaria. However, enhanced severity of anemia was unrelated to either erythropoietic suppression or parasite-driven red blood cell hemolysis. To further explore mechanisms of anemia, circulating inflammatory mediators (IMs) were determined using a 25-plex bead array in P. falciparum-infected (Pf[+]) children (3-36 month, n = 194) stratified into three groups: HIV-1 negative (HIV-1[-]/Pf[+]); HIV-1 exposed (HIV-1[exp]/Pf[+]); and HIV-1 infected (HIV-1[+]/Pf[+]). IL-12, MIG/CXCL9, eotaxin/CCL11, and GM-CSF differed significantly and progressively increased across the groups (HIV-1[-]→HIV-1[exp]→HIV-1[+]). To further explore the relationship between the inflammatory milieu (i.e., cytokines, chemokines, and growth factors) and HIV-1 status, the large panel of IMs was reduced into discrete groups by principal component factor analysis. Of the six principal components that emerged, three components were significantly higher in the HIV-1 [+]/pf[+] and HIV[exp]/Pf[+] groups, demonstrating that inflammatory profiles differ according to HIV-1 status. Additional analyses exploring the relationship between the components and anemia revealed significant positive correlations between Hb and Component 3 (IL-1Ra, IL-7, IL-17, IFN-α, IFN-γ, MIG/CXCL9) in the HIV-1[-]/Pf[+] group, and Component 4 (IL-4, IL-5, IL-12, Eotaxin/CCL11) in HIV-1[+]/Pf[+] children. Further analyses of the HIV-1[+]/Pf[+] group revealed that IL-12 had the strongest association with anemia. Results presented here demonstrate that there are unique relationships between the inflammatory environment and anemia in HIV-1 positive and exposed children with malaria.

Functional haplotypes of Fc gamma (Fcγ) receptor (FcγRIIA and FcγRIIIB) predict risk to repeated episodes of severe malarial anemia and mortality in Kenyan children.

Development of protective immunity against Plasmodium falciparum is partially mediated through binding of malaria-specific IgG to Fc gamma (γ) receptors. Variations in human FcγRIIA-H/R-131 and FcγRIIIB-NA1/NA2 affect differential binding of IgG sub-classes. Since variability in FcγR may play an important role in severe malarial anemia (SMA) pathogenesis by mediating phagocytosis of red blood cells and triggering cytokine production, the relationship between FcγRIIA-H/R131 and FcγRIIIB-NA1/NA2 haplotypes and susceptibility to SMA (Hb < 6.0 g/dL) was investigated in Kenyan children (n = 528) with acute malaria residing in a holoendemic P. falciparum transmission region. In addition, the association between carriage of the haplotypes and repeated episodes of SMA and all-cause mortality were investigated over a 3-year follow-up period. Since variability in FcγR can alter interferon (IFN)-γ production, a mediator of innate and adaptive immune responses, functional associations between the haplotypes and IFN-γ were also explored. During acute malaria, children with SMA had elevated peripheral IFN-γ levels (P = 0.006). Although multivariate logistic regression analyses (controlling for covariates) revealed no associations between the FcγR haplotypes and susceptibility to SMA during acute infection, the FcγRIIA-131H/FcγRIIIB-NA1 haplotype was associated with decreased peripheral IFN-γ (P = 0.046). Longitudinal analyses showed that carriage of the FcγRIIA-131H/FcγRIIIB-NA1 haplotype was associated with reduced risk of SMA (RR 0.65, 95% CI 0.46-0.90; P = 0.012) and all-cause mortality (P = 0.002). In contrast, carriers of the FcγRIIA-131H/FcγRIIIB-NA2 haplotype had increased susceptibility to SMA (RR 1.47, 95% CI 1.06-2.04; P = 0.020). Results here demonstrate that variation in the FcγR gene alters susceptibility to repeated episodes of SMA and mortality, as well as functional changes in IFN-γ production.

Severe malarial anemia: innate immunity and pathogenesis.

Greater than 80% of malaria-related mortality occurs in sub-Saharan Africa due to infections with Plasmodium falciparum. The majority of P. falciparum-related mortality occurs in immune-naïve infants and young children, accounting for 18% of all deaths before five years of age. Clinical manifestations of severe falciparum malaria vary according to transmission intensity and typically present as one or more life-threatening complications, including: hyperparasitemia; hypoglycemia; cerebral malaria; severe malarial anemia (SMA); and respiratory distress. In holoendemic transmission areas, SMA is the primary clinical manifestation of severe childhood malaria, with cerebral malaria occurring only in rare cases. Mortality rates from SMA can exceed 30% in pediatric populations residing in holoendemic transmission areas. Since the vast majority of the morbidity and mortality occurs in immune-naïve African children less than five years of age, with SMA as the primary manifestation of severe disease, this review will focus primarily on the innate immune mechanisms that govern malaria pathogenesis in this group of individuals. The pathophysiological processes that contribute to SMA involve direct and indirect destruction of parasitized and non-parasitized red blood cells (RBCs), inefficient and/or suppression of erythropoiesis, and dyserythropoiesis. While all of these causal etiologies may contribute to reduced hemoglobin (Hb) concentrations in malaria-infected individuals, data from our laboratory and others suggest that SMA in immune-naïve children is characterized by a reduced erythropoietic response. One important cause of impaired erythroid responses in children with SMA is dysregulation in the innate immune response. Phagocytosis of malarial pigment hemozoin (Hz) by monocytes, macrophages, and neutrophils is a central factor for promoting dysregulation in innate inflammatory mediators. As such, the role of P. falciparum-derived Hz (PfHz) in mediating suppression of erythropoiesis through its ability to cause dysregulation in pro- and anti-inflammatory cytokines, growth factors, chemokines, and effector molecules is discussed in detail. An improved understanding of the etiological basis of suppression of erythropoietic responses in children with SMA may offer the much needed therapeutic alternatives for control of this global disease burden.

Functional promoter haplotypes of interleukin-18 condition susceptibility to severe malarial anemia and childhood mortality.

Severe malarial anemia (SMA) is a leading cause of morbidity and mortality in children residing in regions where Plasmodium falciparum transmission is holoendemic. Although largely unexplored in children with SMA, interleukin-18 (IL-18) is important for regulating innate and acquired immunity in inflammatory and infectious diseases. As such, we selected two functional single-nucleotide polymorphisms (SNPs) in the IL-18 promoter (-137G→C [rs187238] and -607C→A [rs1946518]) whose haplotypes encompass significant genetic variation due to the presence of strong linkage disequilibrium among these variants. The relationship between the genotypes/haplotypes, SMA (hemoglobin [Hb], <5.0 g/dl], and longitudinal clinical outcomes were then investigated in Kenyan children (n = 719). Multivariate logistic regression analyses controlling for age, gender, sickle cell trait, glucose-6-phosphate dehydrogenase (G6PD) deficiency, HIV-1, and bacteremia revealed that carriage of the -607AA genotype was associated with protection against SMA (odds ratio [OR] = 0.440 [95% confidence interval {CI} = 0.21 to 0.90], P = 0.031) in children with acute infection. In contrast, carriers of the -137G/-607C (GC) haplotype had increased susceptibility to SMA (OR = 2.050 [95% CI = 1.04 to 4.05], P = 0.039). Measurement of IL-18 gene expression in peripheral blood leukocytes demonstrated that elevated IL-18 transcripts were associated with reduced hemoglobin concentrations (ρ = -0.293, P = 0.010) and that carriers of the "susceptible" GC haplotype had elevated IL-18 transcripts (P = 0.026). Longitudinal investigation of clinical outcomes over a 3-year follow-up period revealed that carriers of the rare CC haplotype (∼1% frequency) had 5.76 times higher mortality than noncarriers (P = 0.001). Results presented here demonstrate that IL-18 promoter haplotypes that condition elevated IL-18 gene products during acute infection are associated with increased risk of SMA. Furthermore, carriage of the rare CC haplotype significantly increases the risk of childhood mortality.

Polymorphic variability in the 3' untranslated region (UTR) of IL12B is associated with susceptibility to severe anaemia in Kenyan children with acute Plasmodium falciparum malaria.

BACKGROUND: Plasmodium falciparum malaria remains a leading cause of morbidity and mortality among African children. Innate immunity provides the first line of defence against P. falciparum infections, particularly in young children that lack naturally-acquired malarial immunity, such as the population examined here. Consistent with the fact that elevated interleukin (IL)-12 is an important component of the innate immune response that provides protective immunity against malaria, we have previously shown that suppression of IL-12 in African children is associated with the development of severe malarial anaemia (SMA). Since the role of IL12B variants in conditioning susceptibility to SMA remains largely unexplored, the association between a single nucleotide polymorphism (1188A→C, rs3212227), SMA (Hb<6.0 g/dL), circulating IL-12p40/p70 levels, and longitudinal clinical outcomes in Kenyan children (n = 756) residing in a holoendemic falciparum malaria transmission area were investigated. RESULTS: Multivariate logistic regression analysis in children with acute malaria (n = 544) demonstrated that carriers of the C allele had increased susceptibility to SMA (CC: OR, 1.674; 95% CI, 1.006-2.673; P = 0.047, and AC: OR, 1.410; 95% CI, 0.953-2.087; P = 0.086) relative to wild type (AA). Although children with SMA had lower IL-12p40/p70 levels than the non-SMA group (P = 0.037), levels did not differ significantly according to genotype. Longitudinal analyses in the entire cohort (n = 756) failed to show any significant relationships between rs3212227 genotypes and either susceptibility to SMA or all-cause mortality throughout the three year follow-up. CONCLUSION: The rs3212227 is a marker of susceptibility to SMA in children with acute disease, but does not appear to mediate functional changes in IL-12 production or longitudinal outcomes during the acquisition of naturally-acquired malarial immunity.

Identification of inflammatory biomarkers for pediatric malarial anemia severity using novel statistical methods.

Areas where Plasmodium falciparum transmission is holoendemic are characterized by high rates of pediatric severe malarial anemia (SMA) and associated mortality. Although the etiology of SMA is complex and multifactorial, perturbations in inflammatory mediator production play an important role in the pathogenic process. As such, the current study focused on identification of inflammatory biomarkers in children with malarial anemia. Febrile children (3 to 30 months of age) presenting at Siaya District Hospital in western Kenya underwent a complete clinical and hematological evaluation. Children with falciparum malaria and no additional identifiable anemia-promoting coinfections were stratified into three groups: uncomplicated malaria (hemoglobin [Hb] levels of ≥11.0 g/dl; n = 31), non-SMA (Hb levels of 6.0 to 10.9 g/dl; n = 37), and SMA (Hb levels of <6.0 g/dl; n = 80). A Luminex hu25-plex array was used to determine potential biomarkers (i.e., interleukin 1ß [IL-1ß], IL-1 receptor antagonist [IL-1Ra], IL-2, IL-2R, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12p70, IL-13, IL-15, IL-17, tumor necrosis factor alpha [TNF-α], alpha interferon [IFN-α], IFN-γ, granulocyte-macrophage colony-stimulating factor [GM-CSF], macrophage inflammatory protein 1 alpha [MIP-1α], MIP-1ß, IFN-inducible protein of 10 kDa [IP-10], monokine induced by IFN-γ [MIG], eotaxin, RANTES, and monocyte chemoattractant protein 1 [MCP-1]) in samples obtained prior to any treatment interventions. To determine the strongest biomarkers of anemia, a parsimonious set of predictor variables for Hb was generated by least-angle regression (LAR) analysis, controlling for the confounding effects of age, gender, glucose-6-phosphate dehydrogenase (G6PD) deficiency, and sickle cell trait, followed by multiple linear regression analyses. IL-12p70 and IFN-γ emerged as positive predictors of Hb, while IL-2R, IL-13, and eotaxin were negatively associated with Hb. The results presented here demonstrate that the IL-12p70/IFN-γ pathway represents a set of biomarkers that predicts elevated Hb levels in children with falciparum malaria, while activation of the IL-13/eotaxin pathway favors more profound anemia.

Clinical predictors of severe malarial anaemia in a holoendemic Plasmodium falciparum transmission area.

Severe malarial anaemia (SMA) is a common complication of Plasmodium falciparum infections, resulting in mortality rates that may exceed 30% in paediatric populations residing in holoendemic transmission areas. One strategy for reducing the morbidity and mortality associated with SMA is to identify clinical predictors that can be readily recognized by caregivers for prompt therapeutic interventions. To determine clinical predictors of SMA, Kenyan children (3-36 months, n = 671) presenting with acute illness at a rural hospital in Siaya District were recruited. Demographic, clinical, laboratory and haematological parameters were measured upon enrolment. As human immunodeficiency virus-1 and bacteraemia promote reduced haemoglobin (Hb) concentrations, children with these infections were excluded from the analyses. Children with P. falciparum mono-infections (n = 355) were stratified into three groups: uncomplicated malaria (Hb >or= 110 g/l); non-SMA (60

Hematological predictors of increased severe anemia in Kenyan children coinfected with Plasmodium falciparum and HIV-1.

Malaria and HIV-1 are coendemic in many developing countries, with anemia being the most common pediatric hematological manifestation of each disease. Anemia is also one of the primary causes of mortality in children monoinfected with either malaria or HIV-1. Although our previous results showed HIV-1(+) children with acute Plasmodium falciparum malaria [Pf(+)] have more profound anemia, potential causes of severe anemia in coinfected children remain unknown. As such, children with P. falciparum malaria (aged 3-36 months, n = 542) from a holoendemic malaria transmission area of western Kenya were stratified into three groups: HIV-1 negative [HIV-1(-)/Pf(+)]; HIV-1 exposed [HIV-1(exp)/Pf(+)]; and HIV-1 infected [HIV-1(+)/Pf(+)]. Comprehensive clinical, parasitological, and hematological measures were determined upon enrollment. Univariate, correlational, and hierarchical regression analyses were used to determine differences among the groups and to define predictors of worsening anemia. HIV-1(+)/Pf(+) children had significantly more malarial pigment-containing neutrophils (PCN), monocytosis, increased severe anemia (Hb < 6.0 g/dL), and nearly 10-fold greater mortality within 3 months of enrollment. Common causes of anemia in malaria-infected children, such as increased parasitemia or reduced erythropoiesis, did not account for worsening anemia in the HIV-1(+)/Pf(+) group nor did carriage of sickle cell trait or G6PD deficiency. Hierarchical multiple regression analysis revealed that more profound anemia was associated with elevated PCM, younger age, and increasing HIV-1 status ([HIV-1(-) --> HIV-1(exp) --> HIV-1(+)]. Thus, malaria/HIV-1 coinfection is characterized by more profound anemia and increased mortality, with acquisition of monocytic pigment having the most detrimental impact on Hb levels.

A novel functional variant in the stem cell growth factor promoter protects against severe malarial anemia.

Plasmodium falciparum malaria is a leading global cause of infectious disease burden. In areas in which P. falciparum transmission is holoendemic, such as western Kenya, severe malarial anemia (SMA) results in high rates of pediatric morbidity and mortality. Although the pathophysiological basis of SMA is multifactorial, we recently discovered that suppression of unexplored hematopoietic growth factors that promote erythroid and myeloid colony development, such as stem cell growth factor (SCGF) (C-type lectin domain family member 11A [CLEC11A]), was associated with enhanced development of SMA and reduced erythropoietic responses. To extend these investigations, the relationships between a novel SCGF promoter variant (-539C/T, rs7246355), SMA (hemoglobin [Hb] < 6.0 g/dl), and reduced erythropoietic responses (reticulocyte production index [RPI], <2.0) were investigated with Kenyan children (n = 486) with falciparum malaria from western Kenya. Circulating SCGF was positively correlated with hemoglobin levels (r = 0.251; P = 0.022) and the reticulocyte production index (RPI) (r = 0.268; P = 0.025). Children with SMA also had lower SCGF levels than those in the non-SMA group (P = 0.005). Multivariate logistic regression analyses controlling for covariates demonstrated that individuals with the homologous T allele were protected against SMA (odds ratio, 0.57; 95% confidence interval [95% CI] 0.34 to 0.94; P = 0.027) relative to CC (wild-type) carriers. Carriers of the TT genotype also had higher SCGF levels in circulation (P = 0.018) and in peripheral blood mononuclear cell culture supernatants (P = 0.041), as well as an elevated RPI (P = 0.005) relative to individuals with the CC genotype. The results presented here demonstrate that homozygous T at -539 in the SCGF promoter is associated with elevated SCGF production, enhanced erythropoiesis, and protection against the development of SMA in children with falciparum malaria.

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.

Suppression of a novel hematopoietic mediator in children with severe malarial anemia.

In areas of holoendemic Plasmodium falciparum transmission, severe malarial anemia (SMA) is a leading cause of pediatric morbidity and mortality. Although many soluble mediators regulate erythropoiesis, it is unclear how these factors contribute to development of SMA. Investigation of novel genes dysregulated in response to malarial pigment (hemozoin [PfHz]) revealed that stem cell growth factor (SCGF; also called C-type lectin domain family member 11A [CLEC11A]), a hematopoietic growth factor important for development of erythroid and myeloid progenitors, was one of the most differentially expressed genes. Additional experiments with cultured peripheral blood mononuclear cells (PBMCs) demonstrated that PfHz decreased SCGF/CLEC11A transcriptional expression in a time-dependent manner. Circulating SCGF levels were then determined for Kenyan children (n = 90; aged 3 to 36 months) presenting at a rural hospital with various severities of malarial anemia. SCGF levels in circulation (P = 0.001) and in cultured PBMCs (P = 0.004) were suppressed in children with SMA. Circulating SCGF also correlated positively with hemoglobin levels (r = 0.241; P = 0.022) and the reticulocyte production index (RPI) (r = 0.280; P = 0.029). In addition, SCGF was decreased in children with reduced erythropoiesis (RPI of <2) (P < 0.001) and in children with elevated levels of naturally acquired monocytic PfHz (P = 0.019). Thus, phagocytosis of PfHz promotes a decrease in SCGF gene products, which may contribute to reduced erythropoiesis in children with SMA.

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.

Haplotypes of IL-10 promoter variants are associated with susceptibility to severe malarial anemia and functional changes in IL-10 production.

Plasmodium falciparum malaria is one of the leading global causes of morbidity and mortality with African children bearing the highest disease burden. Among the various severe disease sequelae common to falciparum malaria, severe malarial anemia (SMA) in pediatric populations accounts for the greatest degree of mortality. Although the patho-physiological basis of SMA remains unclear, dysregulation in inflammatory mediators, such as interleukin (IL)-10, appear to play an important role in determining disease outcomes. Since polymorphic variability in innate immune response genes conditions susceptibility to malaria, the relationship between common IL-10 promoter variants (-1,082A/G, -819T/C, and -592A/C), SMA (Hb < 6.0 g/dL), and circulating inflammatory mediator levels (i.e., IL-10, TNF-alpha, IL-6 and IL-12) were investigated in parasitemic Kenyan children (n = 375) in a holoendemic P. falciparum transmission area. Multivariate logistic regression analyses demonstrated that the -1,082G/-819C/-592C (GCC) haplotype was associated with protection against SMA (OR; 0.68, 95% CI, 0.43-1.05; P = 0.044) and increased IL-10 production (P = 0.029). Although none of the other haplotypes were significantly associated with susceptibility to SMA, individuals with the -1,082A/-819T/-592A (ATA) haplotype had an increased risk of SMA and reduced circulating IL-10 levels (P = 0.042). Additional results revealed that the IL-10:TNF-alpha ratio was higher in the GCC group (P = 0.024) and lower in individuals with the ATA haplotype (P = 0.034), while the IL-10:IL-12 ratio was higher in ATA haplotype (P = 0.006). Results presented here demonstrate that common IL-10 promoter haplotypes condition susceptibility to SMA and functional changes in circulating IL-10, TNF-alpha, and IL-12 levels in children with falciparum malaria.

Polymorphic variability in the interleukin (IL)-1beta promoter conditions susceptibility to severe malarial anemia and functional changes in IL-1beta 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.

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

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.

Suppression of prostaglandin E2 by malaria parasite products and antipyretics promotes overproduction of tumor necrosis factor-alpha: association with the pathogenesis of childhood malarial anemia.

Cytokines and effector molecules are important immunoregulatory molecules in human malaria. Tumor necrosis factor (TNF)-alpha limits malaria parasitemia but also promotes pathogenesis at high concentrations, whereas prostaglandin E2 (PGE2) inhibits TNF-alpha production and is reduced in childhood malaria, at least in part, through suppression of cyclooxygenase (COX)-2 following the ingestion of Plasmodium falciparum hemozoin (pfHz; malarial pigment) by peripheral blood mononuclear cells (PBMCs). Although molecular interactions between TNF-alpha and PGE2 are largely unexplored in human malaria, results presented here show that pfHz-induced suppression of PBMC COX-2 gene products induces overproduction of TNF-alpha. Moreover, addition of exogenous PGE2 to pfHz-treated PBMCs dose-dependently decreased TNF-alpha production, whereas experimental COX inhibitors and antipyretics used during human malaria generated increased TNF-alpha production. Healthy, malaria-exposed children had elevated levels of circulating bicyclo-PGE2/TNF-alpha, compared with children with malarial anemia (P<.01), with systemic bicyclo-PGE2 and TNF-alpha significantly associated with hemoglobin concentrations (r=0.745; P<.01). The results of the present study illustrate that pfHz-induced suppression of PGE2 promotes overproduction of TNF-alpha, which is associated with enhanced malarial anemia.