Background
Malaria is an infectious disease, potentially fatal, caused by Plasmodium protozoan parasites and transmitted by female Anopheles mosquitoes. According to the WHO, malaria is one of the leading causes of death worldwide. Globally, there were an estimated 247 million malaria cases in 2021 in 84 malaria-endemic countries, increasing from 245 million in 2020, with most of this increase coming from countries in the African Region. Furthermore, in 2020, malaria deaths increased by 10% compared with 2019, and between 2019 and 2021, there were 63,000 deaths due to disruptions to essential malaria services during the COVID-19 pandemic. Thus, malaria remains a major public health problem, especially in Africa [
1]. In Senegal, Malaria is endemic throughout, and the entire population is exposed to the disease. In 2020, 0.7% of malaria deaths worldwide occurred in Senegal. The number of malaria cases fell by 4.4% between 2017 and 2020, from 52 to 50 per 1,000 inhabitants at risk, while the number of malaria deaths rose slightly by 1.8% over the same period, from 0.24 to 0.245 per 1,000 inhabitants at risk.
The malaria pathogenesis is complex and needs to be elucidated. During blood-stage infection, the host’s immune system produces proinflammatory cytokines to eliminate the parasite, including IL-6, IFN-γ, and TNF, which are pivotal in controlling the parasite’s growth and elimination. In many studies, the high levels of some pro-inflammatory cytokines have been protective in malaria [
2‐
4]. Pro-inflammatory biomarkers were more elevated in cerebral malaria than in non-cerebral malaria patients [
5]. Regulatory cytokines such as transforming growth factor-β (TGF-β) and IL-10 balance the pro-inflammatory and anti-inflammatory responses. However, in many cases, cytokines have a double role. On the one hand, they contribute to parasitic clearance; on the other, they are responsible for pathological changes encountered in malaria. Cytokine-modulating strategies may represent a promising modern approach to disease management [
3], and the Circulating levels of cytokines have the potential to be biomarkers for severity or protection against malaria.
Recently, the IL-17 proinflammatory cytokine has gained attention among malaria researchers because of its protective role in immunity against extracellular pathogens [
6‐
8] and for the clearance of intracellular pathogens [
9‐
11]. In addition to its essential role in protective immunity, IL-17 is critical in the pathogenesis of various autoimmune inflammatory diseases. IL-17 is a cytokine family that plays a vital role in innate and adaptive immune systems [
12‐
15]. The IL-17 gene is located on chromosome 6p12, comprises three exons and two introns and is coded with six protein members (IL-17A-F). IL-17A is the most essential member of the IL-17 family. The IL-17 receptor family now comprises 5 members (IL-17RA, RB, RC, RD and RE) [
16‐
18].
In mice, it has been demonstrated that elevated IL-17 levels and high IL-4, IL-12α and IFN-γ levels may be a marker of protection against
Plasmodium berghei [
19]. However, the role of IL-17 in human malarial infection outcomes is poorly described, even if increased IL-17 levels in vivax and falciparum malaria and disease severity have been reported [
5,
20]. Further studies are needed to evaluate the implication of IL-17 cytokines' circulation levels and association in malaria protection and/or pathogenesis.
Single Nucleotide Polymorphisms (SNPs) in encoding regions of the
IL-17A gene may influence changes in its expression and, potentially, malaria pathogenesis and risk.
IL-17A SNPs have been linked to several malignancies, including gastric and breast cancer [
21,
22]. However little is known about the association between
IL-17 gene variation and malaria.
To understand the specific IL-17A roles in malaria infection, we conducted this work to analyse IL-17A levels in a retrospective Senegalese cohort, including healthy controls and severe and uncomplicated malaria subjects. Then, the IL-17A gene and its flanking regions were sequenced in individual samples. The SNPs were analysed among individuals concerning malaria disease status to detect their influence on the IL-17A serum levels and its potential associations with malaria severity.
Discussion
Analysis of the genetic effects of inflammatory response gene variants such as inflammatory cytokines is a key step in malaria research to understand the underlying mechanisms of pathogenesis. This knowledge will be essential for identifying specific therapies to prevent mortality or adverse complications associated with severe malaria and the long-term consequences that represent a heavy burden in endemic regions [
30]. It is understood that cytokine gene polymorphisms could affect the serum levels of cytokines by influencing transcriptional regulation. The IL-17 cytokine family is a relatively new family linked to adaptive and innate immune systems. IL-17A are members of the IL-17 cytokine family, essential for the pathogenic activity of IL-17 cells and the production of various proinflammatory mediators in the body [
9,
31]. IL-17A is a multifunctional cytokine which has a protective role in immunity for the clearance of intracellular pathogens such as
Plasmodium spp. [
9‐
11,
19,
32,
33], making it a good therapeutic biomarker in malaria diseases. However, the role of IL-17A in malaria has not yet been extensively investigated. In addition, the association of common polymorphisms with malaria predisposition and cytokine levels was never analysed in Senegal.
Thus, the present study explored IL-17A cytokine levels and gene polymorphism’s influence on IL-17A serum levels. In recent years, evaluating SNPs has been considered a common approach for testing the impact of human genetic variation on diseases [
34].
We have determined the serum IL-17A levels and genotyped
IL-17A variants in Senegalese severe and uncomplicated malaria patients and controls. We observed elevated IL-17A levels in SM patients compared to the UM and healthy cases. Moreover, the high parasitemia in the SM group accompanied the IL-17A increase. This indicates that IL-17A has an essential regulatory role in malaria infection, controlling the intensity of the immune response, as described in the experimental model, as well as human malaria and several other infectious diseases [
5,
19,
35,
36]. IL-17 production is associated with a very high occurrence of chronic inflammation and immunopathological conditions [
31]. Recent data suggest that IL-17 contributes to host protection against diverse infectious organisms during sepsis while inducing hyperinflammation with detrimental outcomes for the host under certain conditions [
37]. Earlier investigations in the experimental model have deciphered the essential role of IL-17. In
P. vivax infection, authors suggest that increasing serum IL-17 levels in malaria patients could be considered a host adaptation mechanism to control changes in blood viscosity, and IL-17 could thus be used as an immunomodulatory agent [
38]. IL-17 appears to act on erythrocytes by remodelling their cell membrane; it is well-known that erythrocytes in malaria are very sensitive to osmotic shock [
38].
We found an elevated level of IL-17A in severe malaria patients who were survivors compared to those who were deceased. Our results seem to confirm the results of Helegbe et al., which showed elevated IL-17 levels together with high IL-4, IL-12α, and IFN-γ levels may be a marker of protection, and the mechanism may be controlled by host factors [
19]. Thus, pro-inflammatory IL-17A cytokine seems to have been protective against fatal malaria. Furthermore, the data agree with the observations of Oyegue-Liabagui et al. [
20], who noted a correlation between Th17 cell count and overall survival in patients with malaria in children.
Immuno-genetic variants are associated with diverse degrees of malaria susceptibility, including cytokine gene polymorphisms that modify their expression and circulating protein levels to reflect inflammatory or anti-inflammatory responses [
39‐
41]. Polymorphisms in the IL-17A cytokine can impact the activity and expression of inflammatory mediators, which can affect interleukin-17 activity [
42,
43].
IL-17A gene polymorphisms have been linked to several malignancies, including gastric and breast cancer [
21,
22]. However little is known about the association between
IL-17 gene variation and malaria. In this study, we performed a genetic analysis of the variations of the
IL-17A gene. We identified 8 SNPs in the
IL-17A gene; among them, 6 SNPs (rs9791323, rs3819024, rs2275913, rs3819025, rs17880588 and rs3748067) were detected with high frequencies (with MAF > 3%) at opposite to 2 other SNPs (rs8193037 and rs8193038) with MAF < 3% were observed. Then, statistical
IL-17A polymorphism analysis was performed using logistic regression to test whether polymorphisms were associated with malaria severity. For the first time, we identified 2 SNPs associated with severe malaria and one associated with uncomplicated malaria. We found that the SNP rs3748067 reduced the risk of severe malaria (odds ratio (OR) = 0.32;
P = 0.04). Instead, the SNP rs3819024 was associated with an increased risk of severe malaria (odds ratio (OR) = 2.61;
P = 0.007). Then, the SNP rs9791323 was associated with a high risk of uncomplicated malaria (odds ratio (OR) = 3.78;
P = 0.045). We
IL-17A rs3819024 G could also be considered a biomarker of malaria severity.
Our data reinforce our knowledge of the genetic variants of the IL-17 cytokine family and their potential roles in malaria. Even though the involvement of
IL-17A variants has yet to be fully elucidated, a previous study had shown that
IL-17F (rs6913472 and rs4715291) and
IL-17RA (rs12159217 and rs41396547) polymorphisms independently modulate susceptibility to Cerebral Malaria and provide evidence that IL-17F protects against it [
44]. The role of SNPs in malaria disease and immunological disorders has been previously reported [
45‐
47]. Associations between cytokine polymorphisms and malaria support that cytokine gene polymorphisms have an unquestionable role in the orchestration of the immune response, leading to different functional scenarios, which in turn influence the outcome of malaria disease establishment and evolution [
20,
48,
49].
The relationship between the
IL-17A polymorphisms and serum IL-17A concentration was analysed. The SNPs rs3748067, rs3819024 and rs9791323 did not show an association with malaria outcome and IL-17A level. Interestingly, we found that the heterozygous rs8193038 AG genotype is significantly associated with higher levels of IL-17A amongst the whole study groups compared to the homozygous rs8193038 AA genotype (OR = 4.9, 95% CI = (2.01- 8.13),
P < 0.001). This data suggests that rs8193038 polymorphism significantly affects
IL-17A gene expression. However, this SNP is located at the intron 1 region, which could correspond to a splicing site and explain our result [
50,
51]. It is well known that the introns regulate gene expression; they contain enhancers or other cis-acting elements that promote the initiation or elongation of transcription. Introns are also involved in alternative splicing and genome imprinting. Intron splicing increases mRNA stability in the nucleus [
52]. It has been shown that intronal SNPs regulate protein synthesis by mRNA splicing [
53]. In addition, functional SNPs in introns are sometimes linked to SNPs in neighbouring genes, influencing mRNA splicing, among other things [
54]. The genome-wide analysis of human SNPs near splice sites revealed 1300 SNPs, which are probably capable of modifying the protein by changing splicing [
55]. Further studies are needed to elucidate how this intronic rs8193038 SNP influences mRNA splicing and IL-17A expression.
Our results fill a gap in the implication of
IL-17A gene polymorphisms on the cytokine level in a Senegalese malaria cohort.
IL-17A gene polymorphisms also may influence cytokine production in response to
Plasmodium infections and may be contribute to the hyperinflammatory responses during severe malaria outcomes. A series of studies performed in the last decade emphasized the
IL-17A SNPs, particularly the rs2275913 variant, and serum cytokines levels in numerous pathogenesis. A recent study by Lang et al. revealed the association between the
IL-17A rs2275913 variant with higher cytokine serum levels and predisposed Preeclampsia development in Chinese patients [
56]. In leprosy, Farag et al
. have demonstrated that IL17A rs2275913 genotype GG was associated with significantly increased IL-17A levels in Egyptian patients [
57]. Finally, another study by Li et al. revealed that
IL-17A polymorphisms may influence hepatocellular carcinoma risk in chronic hepatitis B virus infection via regulating IL-17A production [
58]. However, our study found no association between rs2275913 variant and serum IL-17A levels, even if a high serum level of IL-17A was associated with the heterozygous rs2275913 GA genotype. The lack of significance may be due to a limited number of subjects. These relationships could likely reach statistical significance in a larger cohort of patients. Our results suggest that the association between
IL-17 gene polymorphisms and serum levels may depend on ethnic group populations and/or pathogenesis mechanisms. Then, we found that different IL-17A alleles play different roles in immunity, producing different cytokine levels and disease outcomes, as reported in other studies [
59].
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