Natural monocytic acquisition of haemozoin and rantes polymorphisms: association with malarial disease outcomes and functional changes in children from Western Kenya
Plasmodium falciparum is an important cause of morbidity and mortality in children residing in holoendemic transmission areas largely from severe malarial anaemia (SMA). Although overproduction of inflammatory-derived cytokines are implicated in the immunopathogenesis of severe malaria, the chemokine regulated on activation, normal T-cell expressed and secreted (RANTES, CCL5) is largely unexplored in the context of malaria. Additionally, pro-inflammatory cytokines, such as tumour necrosis factor (TNF)-α, interleukin (IL)-1 and interferon (IFN)- promote increased RANTES production, while anti-inflammatory cytokines (e.g., IL-4, IL-10 and IL-13) down-regulate RANTES biosynthesis. However, the effect of these cytokines on RANTES production in children with malaria anaemia (MA) is poorly understood. Although genetic variation in RANTES is associated with inflammatory, autoimmune and infectious diseases, the role of single nucleotide polymorphisms (SNPs) in conditioning disease outcomes and RANTES production in malaria remains unexplored. These studies investigated the relationship between circulating RANTES with MA severity, thrombocytopaenia, suppression of erythropoiesis, and naturallyacquired haemozoin (pfHz) in monocytes. The functional influence of promoter (403G/A, -4120A/T, and -415]C/T) and intronic (+307A/G) SNPs on malaria outcomes was also examined. These hospital-based cross-sectional studies were performed in infants and young children (age <36mos.) enrolled at Siaya District Hospital, western Kenya. RANTES levels in circulation and from peripheral blood mononuclear cell (PBMC) cultures were measured by a 25-plex cytokine assay and enzyme-linked immunosorbent assay, respectively. Genotyping of -4120A/T, - 4151C/T and +307A/G polymorphisms was performed using Taqman® 5'-allelic discrimination assay, while -403G/A was genotyped by polymerase chain reactionrestriction fragment length polymorphism (PCR-RFLP) method. Results presented here show that suppression of circulating RANTES is associated with increasing severity of MA (p=0.002), decreased erythropoiesis (p=0.049), and malaria-induced thrombocytopaenia (p=0.036). Additional results demonstrate that suppression of circulating RANTES is associated with increasing levels of pigment-containing monocytes (PCM) (p=0.035). In vitro experiments indicate that monocytic acquisition of pfHz is associated with suppression of RANTES from PBMC under both baseline (p=0.001) and stimulated conditions (p=0.072). Although high levels of pfHz in monocytes caused reduced production of IFN- (p=0.003) and IL-10 (p=0.010), multivariate modelling revealed that only PCM (p=0.048, =-0.171) and IL-10 (p<0.0001, =-0.476) were independently associated with RANTES. Subsequent experiments in cultured PBMC from children with naturally-acquired Hz revealed that blockade of endogenous IL-10 caused significant increases in RANTES production (p=0.028). Haplotypic constructs of RANTES (+307/-403/-4120/-4151) revealed higher frequencies of SMA (Hb<5.0g/dL) in AGTC carriers (75.0% vs. 21.5%, p=0.010) and lower prevalence of thrombocytopaenia in AATC carriers (15.4% vs. 84.6%, p=0.036). Multivariate logistic regression analyses illustrated increased susceptibility to SMA (Hb<5.0g/dL) in AGTC carriers (OR, 13.4; 95% CI, 1.3-133.6; p=0.027) and protection from thrombocytopaenia in AATC carriers (OR, 0.2; 95% CI, 0.1-1.0; p=0.05). Moreover, circulating RANTES levels were lower in AGTC (p=0.049), and higher in AATC carriers (p=0.043). Taken together, these results demonstrate that thrombocytopaenia and natural monocytic acquisition of pfHz are a source of reduced RANTES that may contribute to suppression of erythropoiesis in children with MA. Additional results presented here demonstrate that genetic variation in RANTES is important for mediating SMA and thrombocytopaenia during P. falciparum infection. These findings have the potential to contribute to better understanding of SMA pathogenesis, and lead to better disease management modalities and/or vaccine development.