In a gene-based association study with 18 candidate genes for malaria susceptibility using 33 SNPs as genetic markers, this study demonstrated that IL1B, IL4R, IL12RB1 and TNF genes were associated with susceptibility to P. vivax malaria in a population of Pará state, Brazil.
Cytokines are immunomodulatory proteins produced by a wide variety of cells, and with very complex activities. A functional cytokine network is a central element in the homeostasis of the immune response and its alteration may lead to an abnormal immune response. Hence, recent interest has focused upon genes regulating the cytokine expression; in particular on gene polymorphisms that may influence the levels of expression and therefore the overall immune response. Despite evidence demonstrating the importance of IL1B, IL4R, IL12RB1 and TNF genes in P. falciparum malaria pathology, the influence of these variants in P. vivax infections is unknown.
Independent studies reported differences in immune system gene polymorphisms frequencies in distinct malaria-endemic regions . The relevance of these polymorphisms in malaria infections could differ between distinct genetic background populations or etiologic agents, highlighting the importance of studies in different endemic regions. Due to the high admixed nature of the Brazilian population and its substructuring consequences in genetic association studies, this study dealt with this issue with extreme care. The population from Pará state has European, African and Amerindian ancestral groups [16, 21], so that a structured population association test to avoid genetic bias in the analysis was employed to provide reliable results for this specific population.
The IL-1β, IL-12 and the TNF together with IFN-γ are the major cytokines in pro-inflammatory Th1 immune response. IL-1β is predominantly secreted by monocytes and macrophages in initial immune response against infections [22–24] and helps to modulate the expression of IFN-γ and promote the polarization to Th17 immune response in certain circumstances [25, 26]. IL-12 promotes IFN-γ production by T and natural killer (NK) cells and exerts its biological function through binding to the heteromeric interleukin 12 receptor (IL-12R) β1 and β2. The deficiency in IL-12R expression interferes in IL-12 functions and is associated with severe infection in humans [27, 28]. TNF is produced by monocytes and macrophages and its role in malaria pathology was investigated due to reports of high levels of this cytokine in cerebral malaria patients .
IL1B gene was associated with P. falciparum malaria in African populations only [30, 31]. The present study is the first to report the association of -5839C > T SNP promoter with P. vivax malaria susceptibility. The -5839C allele presented a higher frequency in malaria patients then in controls. Despite that, the function of this intronic SNP is not completely elucidated, variability of this important pro-inflammatory gene could represent an important factor in immune regulation. The association of haplotype -31C/-511A in IL1B gene promoter with severe malarial anaemia and circulating IL-1β low levels in children with P. falciparum malaria from Kenya have been shown recently , however, the IL1B -31C > T polymorphism was not associated with cerebral malaria in Thailand . In the present study these two SNPs were not associated with vivax malaria, demonstrating a possible difference in the contribution of these polymorphisms to malaria pathology among populations and parasites. Complementary studies in IL1B gene and IL-1β levels are important to help understand how this gene influences malaria susceptibility and severity.
Recent work in Kenyan patients infected with P. falciparum demonstrated that IL12RB1 rs4229774 and rs383483 polymorphisms were associated with protection against severe malarial anaemia and high parasitaemia levels, but not susceptibility . The present study reports the association of IL12RB1 -1094A/-641C haplotype with P. vivax malaria susceptibility. Despite the lower frequency in the study subjects, this haplotype is present only in the individuals with malaria, suggesting a possible influence in malaria response. The -641C allele leads to a missense variant (i.e. encodes a different amino acid) and can modify the receptor properties and interfere with IL-12 ligation and function. These results suggest that IL12RB1 variants are important in malaria susceptibility and severity. Further studies will be necessary to better understand the IL12RB1 influence on susceptibility and severity in P. vivax malaria.
Polymorphisms in TNF gene promoter have been reported to be associated with symptoms and severity of P. falciparum malaria in different African and Asian populations [35–39]. The present work demonstrated that the TATGG (−1031/-863/-857/-308/-238) TNF haplotype is associated with P. vivax malaria in a Brazilian population. TNF is an important pro-inflammatory cytokine and the TATGG haplotype diverge in two alleles (−1031C and -308A) associated with TNF levels in vivax malaria infection in India . In that work it was hypothesized that -1031C and -308A alleles are rare in Indian malaria patients due to a possible protective effect. The presence of -1031 T and -308 G alleles in TATGG haplotype could be an important factor in vivax malaria susceptibility.
Interleukin 4 receptor (IL-4R), together with IL-4 and IL-13 are important Th2 anti-inflammatory immune response modulators. IL-4R is the principal receptor of these interleukins, and when it is blocked IL-4 and IL-13 function is aborted preventing Th2 immune response modulation . Only a few works investigated IL-4R variants influence on malaria infections. In the present investigation an association of 1902A > G SNP with malaria susceptibility was observed. The 1902 G allele create a missense variant and potentially can modify the receptor properties and interfere with IL-4 and IL-13 functions. It has been shown that the immune response via IL-4, IL4R and IL-13 pathway is important to prevent malaria infection in mice [41, 42]. These studies demonstrated that knockout mice for IL4 and IL4R genes have high resistance to malaria liver stage caused by sporozoites. The absence of modulation mediated by IL-4 to Th2 immune response, the Th1 response mediated by IFN-γ is maintained and promotes a rapid cellular response against sporozoites. The present study shows that 1902A allele is more frequent in malaria patients, although 1902 G presents a higher overall frequency in the study population, 1902A allele could influence the co-regulation between Th1 and Th2 immune response against malaria infections. New complementary studies will be necessary to elucidate the IL4R gene importance in vivax malaria infection.
IL4, IL10, IL12B, IFNG and IFNGR1 genes polymorphisms were associated with symptoms and severity of P. falciparum malaria in studies from African and Asian populations . In the present study, polymorphisms in these genes were not associated with susceptibility to malaria caused by P. vivax. Differences in malaria pathophysiology caused by these two species of parasites could be a possible explanation for the divergences reported. Plasmodium falciparum malaria presents a more acute form of infection reaching more than 50% of erythrocytes leading to cyto-adherence, organ damaged, severe malarial anaemia and cerebral malaria. Malaria caused by P. vivax is characterized by a long incubation period and milder initial symptoms. The parasite infects approximately 2 to 5% of erythrocytes, and can remain dormant in the liver as hypnozoites leading to subsequently relapses . Studies have linked high levels of pro-inflammatory cytokines, such as TNF, IL-1β, IL-6 and IFN-γ in P. falciparum infections [44–47] and TNF, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10 e IL-12 cytokines with P. vivax infections [13, 48–50]. These differences in cytokine profile and gene polymorphisms should reflect a distinct dynamic between the regulatory pathway of pro- and anti-inflammatory cytokines in P. falciparum and P. vivax malaria response, pathology and outcome.