Christopher C. Keller

Elevated Nitric Oxide Production in Children with Malarial Anemia: Hemozoin-Induced Nitric Oxide Synthase Type 2 Transcripts and Nitric Oxide in Blood Mononuclear Cells

ABSTRACT Experiments outlined here investigate the role of nitric oxide (NO) in the pathogenesis of Plasmodium falciparum-induced malarial anemia (MA). The results show that ex vivo and in vitro NO synthase (NOS) activity in peripheral blood mononuclear cells (PBMCs) is significantly elevated in children with MA and inversely associated with hemoglobin levels. Additional experiments using PBMCs from non-malaria-exposed donors demonstrate that physiologic amounts of P. falciparum-derived hemozoin augment NOS type 2 (NOS2) transcripts and NO production. Results of these experiments illustrate that elevated NO production in children with MA is associated with decreased hemoglobin concentrations and that hemozoin can induce NOS2-derived NO formation in cultured blood mononuclear cells.

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.

Acquisition of Hemozoin by Monocytes Down-Regulates Interleukin-12 p40 (IL-12p40) Transcripts and Circulating IL-12p70 through an IL-10-Dependent Mechanism: In Vivo and In Vitro Findings in Severe Malarial Anemia

ABSTRACT Severe malarial anemia (SMA) is a primary cause of morbidity and mortality in immune-naïve infants and young children residing in areas of holoendemic Plasmodium falciparum transmission. Although the immunopathogenesis of SMA is largely undefined, we have previously shown that systemic interleukin-12 (IL-12) production is suppressed during childhood blood-stage malaria. Since IL-10 and tumor necrosis factor alpha (TNF-α) are known to decrease IL-12 synthesis in a number of infectious diseases, altered transcriptional regulation of these inflammatory mediators was investigated as a potential mechanism for IL-12 down-regulation. Ingestion of naturally acquired malarial pigment (hemozoin [PfHz]) by monocytes promoted the overproduction of IL-10 and TNF-α relative to the production of IL-12, which correlated with an enhanced severity of malarial anemia. Experiments with cultured peripheral blood mononuclear cells (PBMC) and CD14+ cells from malaria-naïve donors revealed that physiological concentrations of PfHz suppressed IL-12 and augmented IL-10 and TNF-α by altering the transcriptional kinetics of IL-12p40, IL-10, and TNF-α, respectively. IL-10 neutralizing antibodies, but not TNF-α antibodies, restored PfHz-induced suppression of IL-12. Blockade of IL-10 and the addition of recombinant IL-10 to cultured PBMC from children with SMA confirmed that IL-10 was responsible for malaria-induced suppression of IL-12. Taken together, these results demonstrate that PfHz-induced up-regulation of IL-10 is responsible for the suppression of IL-12 during malaria.

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.

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.

Suppression of Prostaglandin E2 by Malaria Parasite Products and Antipyretics Promotes Overproduction of Tumor Necrosis Factor–α: 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.

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.

A Novel Functional Variant in the Stem Cell Growth Factor Promoter Protects against Severe Malarial Anemia

ABSTRACT 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.

Stage-specific effects of Plasmodium falciparum- derived hemozoin on blood mononuclear cell TNF-α regulation and viral replication

Background:The molecular immunological interactions between HIV and malaria are largely undefined. Since tumor necrosis factor (TNF)-α is elevated during acute malaria and increases with HIV-1 disease progression, TNF-α production may be an important mediator for interactions between malaria and HIV-1. Methods:To examine the stage-specific immunological interactions between HIV and malaria, peripheral blood mononuclear cells (PBMC) and CD14 cells were isolated and cultured from rhesus macaques at different stages of SIV infection. Cultures were stimulated with lipopolysaccharide (LPS) and interferon (IFN)-γ in the presence of Plasmodium falciparum-derived hemozoin (Hz) or synthetic Hz (sHz). TNF-α transcripts and soluble protein were examined by real time reverse transcription-PCR and ELISA, respectively. The effects of Hz on viral replication were determined by measurement of p27 antigen with varying concentrations of TNF-α neutralizing antibodies. Results:Hz and sHz significantly increased LPS- and IFN-γ-induced TNF-α protein and transcripts in PBMC from animals with late stage SIV infection (i.e., AIDS). Hz and sHz also induced high levels of sustained TNF-α transcripts in PBMC from the AIDS group. During the late stage of disease, CD14 cells were the primary source of TNF-α production. Stimulation of PBMC with Hz and sHz significantly increased viral replication that was dose-dependently reduced by the addition of TNF-α neutralizing antibodies. Conclusions:Hz promotes high levels of TNF-α production from PBMC during AIDS and increases viral replication in SIV-infected animals.