Genetic variation in the immune system and malaria susceptibility in infants: a nested case–control study in Nanoro, Burkina Faso

Malaria is a life-threatening infectious disease caused by Plasmodium protozoan parasites and transmitted by Anopheles mosquitoes. Despite global malaria control and elimination efforts, which reduced the number of malaria-related deaths by 50% since 2000, malaria remains a major public health problem, particularly in pregnant women and children from sub-Saharan Africa [1, 2].

Individual risk for malaria infection and disease is complex and multifactorial, and is influenced/modulated by host genetic background [3, 4]. Quantitative genetics have estimated that human genetic factors could explain 25% of individual variation in susceptibility to clinical malaria in Africa [5]. An example are the numerous studies that have demonstrated a prominent role of red blood cell (RBC) polymorphisms, such as haemoglobin-inherited disorders (e.g. thalassaemia, sickle cell disease), erythrocyte membrane protein polymorphisms (e.g. ovalocytosis, spherocytosis, Duffy antigen) and erythrocyte enzymatic disorders (e.g. glucose-6-phosphate dehydrogenase) in malaria susceptibility [6, 7]. On the other hand, there is increasing evidence that identifies polymorphisms in genes related to the immune system as important determinants in susceptibility to malaria infection and disease. Immuno-genetic variants that have been associated with diverse degrees of malaria susceptibility include: (i) polymorphisms in the Human Leucocyte Antigen (HLA), which may affect recognition of parasite antigens [8‐10]; (ii) polymorphisms in cytokine related genes, which may affect protein levels and down-stream functions, such as production of C-reactive protein and immunoglobulin (Ig) isotype switching [11‐16]; (iii) polymorphism in toll-like receptors (TLRs), which may impair the ability of individuals to respond adequately to TLR agonists [17‐21]; and (iv) polymorphisms in IgG Fcγ receptors, which may affect IgG immune complexes binding and the regulation of the IgG subclass production [22‐26].

In Burkina Faso, genetic epidemiology has demonstrated that the wild-type R131 allele (rs1801274) of the FcγRIIA (CD32) and tumour necrosis factor (TNF)-238G allele (rs361525) were associated with protection from clinical malaria and high parasitaemia, respectively, in infants and children until 10 years of age [27‐29]. In contrast, in a family based-study, TNF mutations rs3093664 and rs3093662 were associated with increased risk of parasitaemia and clinical malaria [27]. Overall, most of these studies have been conducted in children and adults, whereas the potential effect of immune genetic variants on infants, who are at great risk of malaria [30] and have particular immunologic characteristics (such as an immature adaptive immune system and the potential protective effect of maternal antibodies and fetal haemoglobin [31‐35]), has not been investigated.

Previous studies by our group in Burkina Faso described that malaria infections and disease during the first year of life is high and has a marked age and seasonal-dependency [30], that individual heterogeneity in the risk of malaria in this age group is strongly influenced by in utero environment, with a profound effect of past placental malaria on fetal immune system [36]. The study hypothesis was that polymorphisms in genes driving Th1/Th2/innate immune response pathways may also affect the development of fetal innate immunity and thus, contribute to the heterogeneity in malaria susceptibility observed during the first year of life [30, 37, 38]. To address this question, 17 single nucleotide polymorphisms (SNPs) in 11 genes of the innate immune system previously associated with malaria-related phenotypes in African populations (including cytokines, TLRs, Fcγ receptors and nitrogen oxide synthase 2, NOS2) [4, 14, 39‐42] were selected to investigated its association with malaria susceptibility in infants living in Nanoro (Burkina Faso), using a nested case–control study design.

This study investigated associations between genetic variants in immune genes and malaria susceptibility in infants in Burkina Faso. The study focused on cytokine, TLR, FcγRIIA/CD32 and NOS2A genes given their key role in innate immune responses against pathogens in early infancy. SNPs in FcγRIIA/CD32 (rs1801274) and in IL-1β (rs1143634) were significantly associated with protection from both malaria infection and clinical disease during the first year of life and FcγRIIA/CD32 SNP rs1801274 genotypes were associated with IL-1RA levels, a cytokine involved in inflammatory responses to malaria infection.

A number of studies have explored the association between FcγRIIA/CD32 rs1801274 SNP and malaria susceptibility resulting in contradictory findings [28, 29, 40, 54‐59]. However, only a limited number included infants and the majority of those focused on severe malaria (e.g. severe malarial anaemia and high-density parasitaemia) [25, 60]. The strongest evidence of FcγRIIA/CD32 rs1801274 (A allele; variant H) protection against blood-stage malaria infections comes from a meta-analysis study on 6928 subjects from Africa and Asia [40]. Results in this study show a similar protective effect of the A allele of the FcγRIIA/CD32 rs1801274 SNP against both malaria infection and disease, with associations confirmed in both co-dominant (AA vs GG and GA vs GG) and dominant (GA/AA vs GG) models after adjusting by potential confounding factors.

Fcγ receptors (FcγRI, FcγRII, FcγRIII) bind to the Fc domain of IgG on immune cells and thereby mediate the initiation of a variety of immunological responses including antigen presentation, phagocytosis, cytotoxicity, release of inflammatory mediators and the modulation of immune responses [61, 62]. Therefore, FcγRs are an important link between cellular and humoral immunity in host defence against malaria infection [62]. FcγRIIA, which is expressed on the surface of all types of cells of the immune system, is a low affinity receptor for monomeric IgG, but binds IgG immune complexes efficiently [63]. FcγRIIA/CD32 rs1801274 has been found to alter the gene function in vitro, by extending the preferential affinity of the wild-type G allele for IgG1 and IgG3 towards affinity for IgG2 [57, 64]. Therefore, it’s reasonable to believe that the efficient binding to IgG2 while retaining its affinity to IgG1 and IgG3 increase the antibody mediated protective effect against malaria. It has been also suggested that differential binding between immune complexes and Fcγ receptors due to polymorphisms in FcγRIIA induce changes in the ability of immune cells to respond to P. falciparum infection through production of inflammatory mediators [58, 62, 64]. Here, the effect of rs1801274 SNP in FcγRIIA/CD32 gene on unstimulated and TLR-stimulated IL-1RA production in cord blood samples was tested, as it has been previously demonstrated that FcγRIIA/CD32 plays a key role in IL-1RA production through binding of immune complexes to monocyte, macrophage and neutrophil lineage cells [52, 53]. Several studies have demonstrated the clinical relevance of IL-1RA in immune responses to malaria [65‐68], which acts modulating disease severity by competing with the pro-inflammatory cytokines IL-1α and IL-1β for binding sites on the IL-1 type I receptor and inhibits IL-1 signalling [69]. While there were no significant differences in the spontaneous production of IL-1RA by cord blood cells across FcγRIIA/CD32 rs1801274 genotypes, upon TLR7/8 stimulation, individuals with GA genotype had significantly higher levels of IL-1RA compared with those with the wild type GG genotype. Remarkably, in infants from this cohort TLR7/8-induced IL-1RA production in cord blood was an independent predictor of malaria protection during the first year of life [36]. Therefore, study findings support the hypothesis that variability in susceptibility to malaria across FcγRIIA/CD32 rs1801274 genotypes is mediated, at least partially, by changes in immune complex-mediated cytokine production. In addition to the protective effect of IgG1 and IgG3, FcgR2A mutant carriers are likely to be protected due to the high affinity of the A allele product to IgG2 possibly by improving the antibody mediated protective effect. Given that this protective effect could be effective through the mediation of a variety of immunological responses including inflammatory mediators, level of IL1-RA could represent one of the changes in innate immune response to malaria infection between the mutant A and the wild-type G allele carriers. The fact that a significant difference was not observed for homozygous allele could be due to limited number of individuals carrying the AA genotype in the subset of data analysed.

The second genetic variant in immune genes associated with malaria protection in this study was the IL-1β rs1143634 SNP. The carriage of IL-1β rs1143634 mutated allele A (both in homozygosity and heterozygosity) was associated with a decreased risk of malaria infection (84% and 43% reduction, respectively). In addition, AA homozygote carriers (but not AG) had a significantly reduced risk of clinical malaria, suggesting an enhanced protective effect of AA homozygosity. This is the first study to report a protective effect of IL-1β rs1143634 SNP against malaria infection and uncomplicated disease in infants. Previous studies assessing associations of IL-1β rs1143634 and malaria susceptibility reported heterogeneous findings in children including an association with higher peripheral parasitaemia in Ghanaian children (n = 461; aged 1–12 years) [70], an association with severe malaria in Gambian children (n = 1420; aged < 5 years) [71], or no significant association with malaria susceptibility in Cameroonian children (n = 1862; aged 1–14 years) [72]. These contrasting results may be attributed to differences in age groups, ethnicity and/or malaria phenotype evaluated.

IL-1β is a pro-inflammatory cytokine that is implicated in the first line of defence against pathogens, including the hepatic and erythrocytic stages of malaria parasites [73‐75]. However, high levels of sustained IL-1β production may induce pathogenic effects that promote disease manifestation and severity [76‐78]. Previous studies have already demonstrated a correlation between IL-1β rs1143634 SNP and increased in vitro production of IL-1β [79, 80]. To investigate whether the observed protective effect of the rs1143634 SNP in this study was related to functional changes in IL-1β production, IL-1β levels in cord blood were compared across genotypic groups but no significant differences were observed neither in un-stimulated or TLR7/8-stimulated samples. Genetic variations occurring in genes encoding inflammatory cytokines can have a direct effect on the innate immune responses to malaria infection and disease manifestation. In this regard, the protective effect of the IL-1β rs1143634 could be due to the modulation of IL-1β level towards a protective effect instead to levels that could be harmful.

However, these findings should be interpreted with caution due to very low number of individuals carrying the AA genotype among the subset of infants that could be analysed (n = 4) and, therefore, potential functional changes in IL-1β production by IL-1β rs1143634 SNP cannot be ruled out. Likewise, it is worth noting that IL-1β production is likely to be influenced by other SNPs in IL-1β gene, as well as polymorphisms in inflammatory mediators such as TNF [81‐84] that were not assessed in this study. Correlation between SNP of interest and cytokine levels in different genotypes should be further explored in different malaria phenotypes (severe, uncomplicated, infection) in the field to elucidate these contrasting results.

Other genes coding for major TLRs (i.e. TLR1, 4 and 9) and cytokines (i.e. IL-4, IL-10, IL-17F and TNF) did not show significant association with malaria infection and clinical episodes, although rs1800890 SNPs in IL-10 gene showed a trend towards increased susceptibility to malaria infection or clinical episode in multivariate analysis (P = 0.057 and P = 0.052, respectively). IL-10 is a key immuno-regulator of immunity to infections [85] and has been associated with various malaria-related phenotypes, such as uncomplicated malaria, severe malaria, severe malaria anaemia and high parasite density [86‐91]. In addition, a number of studies have reported associations between IL-10 SNPs and susceptibility to malaria with either protective effects in some SNPs and increased susceptibility in others [72, 92‐95]. Therefore, this tendency of an increased risk of malaria among carriers of rs1800890 SNP in IL-10 gene, could results from changes that do not allow adequate modulation of inflammatory responses since IL-10 is an anti-inflammatory cytokine.

This study showed that the mutated A allele of IL-1β rs1143634 and mutated A allele of FcγRIIA/CD32 rs1801274 are associated with protection against both malaria infections and uncomplicated disease during the first year of life. These findings suggest that genetic polymorphisms in genes driving innate immune responses could condition malaria susceptibility during the first year of life, possibly by modulating production of inflammatory mediators. Future investigations on the epistatic effects of immune genes polymorphisms described here and functional studies may contribute to further understand how host genetic variation influences malaria susceptibility in infancy.