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

Abstract

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.

FIG. 1.

Effect of PfHz on SCGF gene expression. (A) Gene expression profiles of SCGF in PBMCs (1 × 10⁶ cells/ml) from healthy, malaria-naïve U.S. donors (n = 5) stimulated with PfHz (10 μg/ml). Gene expression profiles were determined for pooled samples from five individuals by use of a cDNA membrane array. Densitometry readings (arbitrary units [AU]) were obtained, and data were normalized to a panel of housekeeping genes. Values are presented as means. IFN-γ, gamma interferon. (B) Temporal effect of PfHz and sHz on SCGF transcriptional expression in cultured PBMCs from healthy, malaria-naïve U.S. donors (n = 6). SCGF mRNA was quantified by real-time RT-PCR. PBMCs (1 × 10⁶ cells/ml) were cultured in medium alone (solid gray line; controls) and following stimulation with PfHz (10 μg/ml; solid black line) or sHz (10 μg/ml; dashed gray line) for 48 h. Values are presented as means ± standard errors of the means. Statistical significance was determined by Student's t test. *, P < 0.05 compared to unstimulated conditions.

FIG. 2.

SCGF production in children with malaria. Plasma was obtained from healthy, aparasitemic children and children with acute malaria, and SCGF concentrations were determined by ELISA. (A) Children were categorized according to Hb concentrations, as defined in Materials and Methods. Clinical groups were HC (n = 18), UM (n = 11), MA (n = 45), and SMA (n = 16). Each box represents the interquartile range, the line through the box is the median, whiskers show the 10th and 90th percentiles, and circles are outliers. Statistical significance was determined by the Kruskal-Wallis test, followed by post hoc comparisons using the Mann-Whitney U test. (B) PBMCs were isolated from peripheral blood (<3.0 ml) of children with various degrees of malarial anemia and cultured (1 × 10⁶ cells/ml) under baseline conditions. Children were categorized according to Hb concentrations, as defined in Materials and Methods. Clinical groups were HC (n = 6), MA (n = 37), and SMA (n = 12). Supernatants were obtained at 48 h for SCGF determination by ELISA. Data are presented as means ± standard errors of the means. Statistical significance was determined by Student's t test.

FIG. 3.

Association of circulating SCGF with anemia outcomes. Plasma was obtained from healthy, aparasitemic children and children with acute malaria, and SCGF concentrations were determined by ELISA. (A) Correlation between SCGF concentrations and Hb levels for all clinical groups (n = 90). (B) Association between SCGF and RPI for anemic children (n = 61). Relationships between SCGF, Hb, and RPI were determined by Spearman's correlation coefficient. (C) Circulating SCGF levels in anemic children (n = 48) were stratified according to RPI (RPIs of <2 versus RPIs of ≥3). Each box represents the interquartile range, the line through the box is the median, whiskers show 10th and 90th percentiles, and circles are outliers. Statistical significance was determined by the Mann-Whitney U test.

FIG. 4.

Circulating SCGF levels in children with various levels of intramonocytic PfHz. Circulating concentrations of SCGF were determined for children with various degrees of malarial anemia and stratified according to the percentage of PCM (none [0%], n = 52; low [≤10%], n = 13; and high [>10%], n = 7). Each box represents the interquartile range, the line through the box is the median, whiskers show 10th and 90th percentiles, and circles are outliers. Statistical significance was determined by the Kruskal-Wallis test, followed by post hoc comparisons using the Mann-Whitney U test.