# An elevated level of interleukin-17A in a Senegalese malaria cohort is associated with rs8193038 IL-17A genetic variant

Fatou Thiam, Gora Diop, Cédric Coulonges, Celine Derbois, Alassane Thiam, Abou Abdallah Malick Diouara, Mame Ndew Mbaye, Mamadou Diop, Cheikh Momar Nguer, Yakhya Dieye, et al.

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## To cite this version:

Fatou Thiam, Gora Diop, Cédric Coulonges, Celine Derbois, Alassane Thiam, et al. An elevated level of interleukin-17A in a Senegalese malaria cohort is associated with rs8193038 IL-17A genetic variant. BMC Infectious Diseases, 2024, 24, pp.275. 10.1186/s12879-024-09149-8. hal-04905298

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# An elevated level of interleukin-17A in a Senegalese malaria cohort is associated with rs8193038 IL-17A genetic variant

Fatou Thiam¹*, Gora Diop²,³, Cedric Coulonges⁴, Celine Derbois⁵, Alassane Thiam³,
Abou Abdallah Malick Diouara¹, Mame Ndew Mbaye¹, Mamadou Diop¹, Cheikh Momar Nguer¹,
Yakhya Dieye¹,⁶, Babacar Mbengue⁷, Jean-Francois Zagury⁴, Jean-Francois Deleuze⁵ and Alioune Dieye⁷

## Abstract

Malaria infection is a multifactorial disease partly modulated by host immuno-genetic factors. Recent evidence has demonstrated the importance of Interleukin-17 family proinflammatory cytokines and their genetic variants in host immunity. However, limited knowledge exists about their role in parasitic infections such as malaria. We aimed to investigate IL-17A serum levels in patients with severe and uncomplicated malaria and gene polymorphism's influence on the IL-17A serum levels. In this research, 125 severe (SM) and uncomplicated (UM) malaria patients and 48 free malaria controls were enrolled. IL-17A serum levels were measured with ELISA. PCR and DNA sequencing were used to assess host genetic polymorphisms in *IL-17A*. We performed a multivariate regression to estimate the impact of human *IL-17A* variants on IL-17A serum levels and malaria outcomes. Elevated serum IL-17A levels accompanied by increased parasitemia were found in SM patients compared to UM and controls (P < 0.0001). Also, the IL-17A levels were lower in SM patients who were deceased than in those who survived. In addition, the minor allele frequencies (MAF) of two IL-17A polymorphisms (rs3819024 and rs3748067) were more prevalent in SM patients than UM patients, indicating an essential role in SM. Interestingly, the heterozygous rs8193038 AG genotype was significantly associated with higher levels of IL-17A than the homozygous wild type (AA). According to our results, it can be concluded that the IL-17A gene rs8193038 polymorphism significantly affects IL-17A gene expression. Our results fill a gap in the implication of *IL-17A* gene polymorphisms on the cytokine level in a malaria cohort. IL-17A gene polymorphisms also may influence cytokine production in response to Plasmodium infections and may contribute to the hyperinflammatory responses during severe malaria outcomes.

**Keywords** Plasmodium falciparum, Cytokines, Single nucleotide polymorphism, Severe malaria

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BMC

© The Author(s) 2024. **Open Access** This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

<footnote>*Correspondence:
Fatou Thiam
fatou54.thiam@ucad.edu.sn
Full list of author information is available at the end of the article</footnote>
## 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.

## Methods

### Study participants

Our study was conducted in Senegal, a malaria-endemic country in the Sahelian zone of West Africa. Malaria patients were enrolled using a retrospective cohort from the Hospital Principal of Dakar between the 2012–2015 period and corresponded to subjects with Plasmodium-positive Quantitative Buffy Coat (QBC) [23, 24]. These patients were classified into Uncomplicated Malaria (UM) and Severe Malaria (SM) groups, according to the criteria defined by Saissy et al. 2003 and previously described in [25]. The inclusion criteria for these patients were: firstly, black Senegalese individuals with *P. falciparum* infection confirmed in diagnosis; secondly, persons born in Senegal whose parents and grandparents were born in Senegal; and finally, individuals who have not travelled in the last three months of their hospital admission. The healthy control subjects corresponded to the exposed and uninfected subjects group in the same areas and inclusion criteria. They correspond to patients visiting the hospital for annual health check-up with a Plasmodium-negative QBC test.
The study protocol was approved by UCAD's Committee on Research and Ethics (CER), which considers that the research proposed respects the appropriate ethical standard and, as a result, approves its execution under "Protocole 0344/2018/CER-UCAD". Written informed consent was obtained from adult participants and parents or legal representatives of children.

The study included 48 CTR, 54 UM and 71 SM subjects. The study's objectives have been explained clearly using the local dialect before including patients in hospital centres.

## Serum collection and IL-17A quantification

Blood samples were collected from malaria patients and controls and drawn into EDTA vacutainer tubes. Samples were centrifuged, plasma aliquoted, and stored at -20 °C until testing. Sandwich quantified serum levels of IL-17A enzyme-linked immunosorbent assays (ELISA) using the pre-designed kit (Elabscience Biotechnology®, USA) as per the manufacturer. The sensitivity of the assay protocol was 2.38 pg/mL, and the coefficient of variation (CV) for intra-assays and inter-assays was < 8% and < 10%, respectively. The micro ELISA plates provided in the kit have been pre-coated with an antibody specific to Human IL-17A. Standards and samples were added to the micro ELISA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Human IL-17A and Avidin-Horseradish Peroxidase (HRP) conjugate were added successively to each microplate well and incubated. The wells were washed 3 times with 350 uL of wash buffer, and the substrate solution was added to each well. Only those wells that contain Human IL-17A, biotinylated detection antibody and Avidin-HRP conjugate will have appeared blue in colour. The enzyme-substrate reaction was terminated by adding a stop solution, and the colour turned yellow. The optical density (OD) was measured spectrophotometrically at

a wavelength of 450 nm ± 2 nm. The OD value was proportional to the concentration of Human IL-17A. We have calculated the concentration of Human IL-17A in the samples by comparing the OD of the samples to the standard curve. The sensitivity of the assay protocol was 18.75 pg/mL, and the coefficient of variation (CV) for intra-assays and inter-assays was < 10%.

## Genotyping

DNA was extracted from the peripheral blood of each subject by using standard Qiagen Kits according to the manufacturer's recommendations. The concentration and quality of the extracted DNA were measured using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The IL-17A polymorphisms were genotyped using the polymerase chain reaction method. The Oligonucleotide primers used to amplify promoters and exon regions are listed in Table 1. The PCR reactions were performed using a Gotaq® Green Master Mix (Promega, Germany) in a total volume of 25 µl containing 25 ng of genomic DNA (5 ng/µl) and 2.5 µL of each primer (10 µM). The PCR conditions were initial denaturation at 95 °C for 5 min, 35 cycles at 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 5 min, with a final extension at 72 °C for 10 min. The amplicons were purified using BioGel P100 gels (Bio-Rad). Sequencing reactions (2 µL of PCR product) were performed using the dye terminator v3.1 method in an ABI PRISMs 3730 DNA Analyzer (Applied Biosystems, Foster City, CA, USA). Sequencing conditions were: 96 °C for 5 min, 25 cycles of 96 °C for 10 s, 60 °C for 4 min and 15 °C forever, and PCR products were purified with Sephadex G50 superfine columns (GE Healthcare). Alignment of acquired sequences and SNP discovery were performed using NC_000006.12 as a reference. Analysis was performed with Genalys version 2.0b software [26].

<table><caption>**Table 1** List of Primers used to amplify the exons of IL17A by polymerase chain reaction (PCR)</caption><thead><tr><td>PRIMERS</td><td>Sequence (5' &gt; 3')</td><td>Primer Orientation</td><td>Product Lenght (bp)</td></tr></thead><tbody><tr><td>IL17APromPF</td><td>CCAAGTTGCTTGGTAGCATG</td><td>Sense</td><td>557</td></tr><tr><td>IL17APromPR</td><td>CAGTGGGTTCAGGGGTGACA</td><td>Antisense</td><td></td></tr><tr><td>IL17AEx1PF</td><td>TAGCAGCTCTGCTCAGCTTC</td><td>Sense</td><td>563</td></tr><tr><td>IL17AEx1PR</td><td>CTTCTTGTGTGGTTTAGCCC</td><td>Antisense</td><td></td></tr><tr><td>IL17AEx3_1PF</td><td>TTGGTCTTCTTCTGTCTGTC</td><td>Sense</td><td>291</td></tr><tr><td>IL17AEx3_1PR</td><td>AGTCAAACCTTCCTTCTTGG</td><td>Antisense</td><td></td></tr><tr><td>IL17AEx3_3PF</td><td>ATAATGGCCCTGAGGAATGG</td><td>Sense</td><td>554</td></tr><tr><td>IL17AEx3_3PR</td><td>ACCCCTGGATTTGGAATAGG</td><td>Antisense</td><td></td></tr></tbody></table>

PF Primer Forward, PR Primer Reverse, bp base pair
## Statistical analysis

GraphPad Prism v9.5.1 was employed for all statistical analyses. One-way variance analysis ANOVA or Student statistical tests compared serum levels of IL-17A in malaria groups and controls. We calculated Allelic frequencies and Hardy-Weinberg equilibrium, as described previously (Rodriguez, Gaunt & Day, 2009) [27]. Then, as reported previously, the differences in allelic frequencies between the three groups (SM, UM, CTR) were determined using the logistic regression analysis method [28, 29]. Next, associations between IL-17A genotypes and serum levels and malaria outcomes were performed using the Mann-Whitney test, and then associations with *P* values < 0.05 were considered statistically significant.

## Results

### Characteristics of malaria patients and healthy controls

A total of 125 malaria patients and 48 controls were included in this retrospective study. The clinical characteristics of the malaria and control groups are summarized in Table 2. The mean values of all haematological parameters included in our study were determined in three categories of enrolled subjects, and the statistical analysis was done using an ANOVA test. A significant difference was observed in parameters while comparing malaria cases and controls, such as age, haemoglobin level, blood cell parameters and leukocyte cells. However, there were no statistically significant differences in monocytes, Basophils. We then compared the same

parameters between severe and uncomplicated malaria patients using the Student's t-test. We found the same results as above except for lymphocytes and neutrophils, for which the differences in means between SM and UM were insignificant.

### Severe malaria patients displayed higher serum IL-17A compared to uncomplicated malaria and controls

ELISA test quantified the level of IL-17A. As shown in Fig. 1, the mean level of IL-17A was significantly higher in the SM group (mean ± SE: 37.74 ± 140 pg/mL) compared to that of the UM and control groups (mean ± SD: 1.69 ± 2.4 pg/mL and 0.67 ± 1.07 pg/mL, respectively; *P* < 0.001) (Fig. 1a). In the SM group, we observed a lower level of IL-17A in patients who were deceased compared to those who survived (mean ± SD: 25.92 ± 46.48 and 40.78 ± 150.67 pg/mL, respectively). Still, the difference was not significant (*P* = 0.56); the lack of significance may be partly due to the small sample size (Fig. 1b). The parasitemia at the time of diagnosis in SM patients was higher than that of the UM patients (mean ± SD: 9039.54 ± 17891 and 3084 ± 14092, respectively; *P* = 0.0022). In addition, there was a decrease in parasitemia in the deceased compared to the survivors, but the difference was not significant (mean ± SD: 9915.54 ± 19185 and 6496 ± 7161, respectively; *P* = 0.8) (Fig. 1c, d).

<table><caption>**Table 2** Baseline characteristics of malaria patients and healthy controls. The means of selected parameters were compared in three groups using the ANOVA test (*P* value<sup>a</sup>) or in two groups, SM and UM using the Student test (*P* value<sup>b</sup>). Values were statistically significant when *p* < 0.05</caption><thead><tr><th>Characteristics</th><th>CTR (n = 48)</th><th>UM (n = 54)</th><th>SM (n = 71)</th><th>P value<sup>a</sup></th><th>P value<sup>b</sup></th></tr></thead><tbody><tr><td>Age, (M ± SD, year)</td><td>30.54 (16.14)</td><td>13.72 (17.56)</td><td>20.76 (20.17)</td><td>P < 0.0001</td><td>0.07</td></tr><tr><td>Gender, (%)</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Male</td><td>21 (43.75)</td><td>19 (35.20)</td><td>37 (52.11)</td><td></td><td></td></tr><tr><td>Female</td><td>27 (56.25)</td><td>31 (57.40)</td><td>31 (43.67)</td><td></td><td></td></tr><tr><td>Hb (M ± SD, g/dl)</td><td>12.95 (1.930)</td><td>12.49 (2.855)</td><td>8.631 (3.150)</td><td>P < 0.0001</td><td>P < 0.0001</td></tr><tr><td>Hematocrit (M ± SD, %)</td><td>38.76 (5.762)</td><td>37.65 (8.382)</td><td>25.22 (9.631)</td><td>P < 0.0001</td><td>P < 0.0001</td></tr><tr><td>MCV (M ± SD, fL)</td><td>83.25 (5.687)</td><td>88.60 (12.90)</td><td>79.14 (13.25)</td><td>P = 0.0009</td><td>P = 0.007</td></tr><tr><td>RBCs (M ± SD, × 10<sup>6</sup>/µL)</td><td>4.699 (0.632)</td><td>4.145 (0.796)</td><td>3.149 (1.162)</td><td>P < 0.0001</td><td>P < 0.0001</td></tr><tr><td>MCHC (M ± SD, pg/cell)</td><td>33.60 (0.626)</td><td>33.74 (3.159)</td><td>33.33 (1.536)</td><td>P = 0.6685</td><td>P = 0.289</td></tr><tr><td>Leucocyte (M ± SD, × 10<sup>3</sup>/µL)</td><td>5.933 (2.405)</td><td>8.217 (4.351)</td><td>12.40 (6.952)</td><td>P < 0.0001</td><td>P = 0.0008</td></tr><tr><td>Neutrophil (M ± SD, %)</td><td>43.71 (10.64)</td><td>59.95 (15.36)</td><td>54.05 (25.60)</td><td>P = 0.0068</td><td>P = 0.766</td></tr><tr><td>Lymphocyte (M ± SD, %)</td><td>43.21 (10.89)</td><td>29.27 (13.13)</td><td>22.86 (17.03)</td><td>P < 0.0001</td><td>P = 0.538</td></tr><tr><td>Monocyte (M ± SD, %)</td><td>7.970 (2.887)</td><td>7.312 (5.597)</td><td>6.662 (4.691)</td><td>P = 0.5191</td><td>P = 0.663</td></tr><tr><td>Eosinophil (M ± SD, %)</td><td>4.210 (3.829)</td><td>3.2 (2.815)</td><td>0.461 (0.451)</td><td>P < 0.0001</td><td>P = 0.0002</td></tr><tr><td>Basophil (M ± SD, %)</td><td>0.8925 (0.558)</td><td>0.717 (0.575)</td><td>0.6858 (0.729)</td><td>P = 0.3369</td><td>P = 0.239</td></tr></tbody></table>

M ± SD mean ± Standard Deviation, CTR Control, UM Uncomplicated Malaria, SM Severe Malaria, Hb Hemoglobin, MCV Mean Corpuscular Volume, RBCs Red Blood Cells, MCHC Mean Corpuscular Hemoglobin Concentration
<figure><img src="image_5.png" /><figcaption>**Fig. 1** Parasite density and IL17A levels in different categories of enrolled subjects. a, b Serum level IL-17 between SM, UM and Control group and survivors and deceased SM patients. c, d Parasitemia between SM, UM and Control group, survivors, and deceased SM patients. Data representing mean pg/ml ± SE for IL17A and mean P/uL for parasitemia were analysed with one way ANOVA or Student Test for comparison. *P* < 0.05 was considered statistically significant</figcaption></figure>

Distribution of IL-17A polymorphisms and association
with the risk of severe malaria outcome

We analyzed genetic variations on the *IL-17A* gene
in the promoter region and all the coding regions by
sequencing and identified eight SNPs, including 4 SNPs
located in the 5'UTR: *IL-17A* + 521A/C (rs9791323),
*IL-17A* + 606A/G (rs3819024), *IL-17A* + 849G/A
(rs2275913), *IL-17A* + 973G/A (rs8193037); two SNP
located within intron 1: *IL-17A* + 1090G/A (rs3819025),
*IL-17A* + 1198A/G (rs8193038); one located within exon
3: *IL-17A* + 3840G/A (rs17880588) and one located in the
3'UTR: *IL-17A* + 5151C/T (rs3748067) (Fig. 2).

The Minor Allele frequencies (MAF) of the eight
SNPs loci of the *IL-17A* gene were identified using the
Hardy-Weinberg equilibrium test (Table 3). Among
them, 6 SNP (rs9791323, rs3819024, rs2275913,

rs3819025, rs17880588 and rs3748067) were detected
with high frequencies (with MAF > 3%), unlike 2 other
SNPs (rs8193037 and rs8193038) with MAF < 3% were
observed (Table 3). First, comparisons were performed
among the three groups to test whether polymorphisms
were associated with malaria severity. Then, statisti-
cal *IL-17A* polymorphisms analysis was performed
using logistic regression tests with an adjustment for
potential confounders such as Hb polymorphisms. The
SNPS rs3819024 and rs3748067 yielded a significant
association with Severe Malaria. For SM vs UM, the
*P* = 0.007 (OR 2.61, 95% CI 0.35– 0.91) and *P* = 0.04 (OR
0.32, 95% CI 1–2.1), respectively. In addition, the SNP
rs9791323 was associated with uncomplicated malaria
*P* = 0.045 (OR 3.78, 95% CI 2.13 – 7.8) with higher MAF
in UM (11.3%) patients compared to controls (3.3%).
<figure><img src="image_6.png" /><figcaption>**Fig. 2** Mapping of Interleukin-17A (IL-17A) gene and the polymorphisms genotyped. Schema of the gene encoding Interleukin 17A (IL17A) (a) The human IL17A gene (located at 6p12-2) contains 3 exons that span 4 kb. Exons are described by number. AUG: the transcription start site. Black arrows mark eight specific polymorphisms identified in the IL-17A gene</figcaption></figure>

<table><caption>**Table 3** Minor Allele Frequencies of single nucleotide polymorphism (SNP) of IL17-A gene and association analysis with severe outcomes. The *p* values for statistical tests were performed using each polymorphism's linear regression model analysis. Association analysis of IL-17A was served separately, and corrections to each other were applied to reflect the real effect of each. Analysis has been carried out by comparing SM vs UM, UM vs CTR, and SM vs CTR. Borderline (0.05 ≤ *p* ≤ 0.1) and significant (0 – *p* ≤ 0.05) *p* values are in bold. *The OR (odds ratio) and CI (Confidence intervals) were shown when *p* values were significant for malaria (SM)</caption><thead><tr><th rowspan="2">SNP</th><th rowspan="2">Gene location</th><th rowspan="2">NCBI dbSNP number</th><th colspan="4">MAF</th><th colspan="3">HWE, exact P-value</th></tr><tr><th>CTR</th><th>UM</th><th>SM</th><th>Global</th><th>SM vs UM</th><th>UM vs CTR</th><th>SM vs CTR</th></tr></thead><tbody><tr><td>+521 (A&gt;C)</td><td>5 Prime UTR</td><td>rs9791323</td><td>0.033</td><td>0.113</td><td>0.075</td><td>0.075</td><td>0.37</td><td><strong>0.045</strong><br>3.78 (2.13 – 7.8)*</td><td>0.2477</td></tr><tr><td>+606 (A&gt;G)</td><td>5 Prime UTR</td><td>rs3819024</td><td>0.239</td><td>0.113</td><td>0.25</td><td>0.203</td><td><strong>0.007</strong><br>2.61 (1.16 – 5.21)*</td><td>0.02</td><td>0.876</td></tr><tr><td>+849 (G&gt;A)</td><td>5 Prime UTR</td><td>rs2275913</td><td>0.053</td><td>0.028</td><td>0.079</td><td>0.055</td><td>0.15</td><td>0.48</td><td>0.59</td></tr><tr><td>+973 (G&gt;A)</td><td>5 Prime UTR</td><td>rs8193037</td><td>0.021</td><td>0.009</td><td>0.024</td><td>0.018</td><td>0.63</td><td>0.60</td><td>1</td></tr><tr><td>+1090 (G&gt;A)</td><td>intronlef</td><td>rs3819025</td><td>0.085</td><td>0.104</td><td>0.095</td><td>0.096</td><td>0.83</td><td>0.81</td><td>1</td></tr><tr><td>+1198 (A&gt;G)</td><td>intronlef</td><td>rs8193038</td><td>0.021</td><td>0.009</td><td>0.024</td><td>0.018</td><td>0.63</td><td>0.60</td><td>1</td></tr><tr><td>+3840 (G&gt;A)</td><td>coding_sequence</td><td>rs17880588</td><td>0.053</td><td>0.034</td><td>0.043</td><td>0.043</td><td>1</td><td>0.67</td><td>1</td></tr><tr><td>+5151 (C&gt;T)</td><td>3 Prime UTR Variant</td><td>rs3748067</td><td>0.125</td><td>0.055</td><td>0.155</td><td>0.112</td><td><strong>0.04</strong><br>0.32 (0.15 – 0.90)*</td><td>0.10</td><td>0.54</td></tr></tbody></table>

SM Severe Malaria, UM Uncomplicated Malaria, CTR Control group, MAF Minor Allele Frequency, HWE Hardy-Weinberg

## Association of SNP IL-17A + 1198 A/G (rs8193038)) and serum IL-17A concentration

The relationship between the IL-17A polymorphisms and serum IL-17A concentration was analysed regardless of the differences in the study groups. We compared IL17A levels between the different genotypes of the polymorphisms analysed in the study and found differences in levels depending on genotype. We found that some

genotypes were associated with increased IL17A levels (Fig. 3c, f, h), while the opposite was true for others (Fig. 3a, b, d, e, g).

However, only 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).
<figure><img src="image_7.png" /><figcaption>**Fig. 3** Concentration of IL-17A according to its genotypes. ELISA quantified serum levels of IL-17A in a total of 173 subjects comprising Severe and Uncomplicated malaria subjects and Controls. Mean IL-17A levels in the different genotypes of rs2275913 (a) rs3819024 (b) rs2275913 (c) rs81933037 (d) rs3819025 (e) rs8193038 (f) rs17880588 (g) rs3748067 (h) Polymorphisms were compared by ANOVA followed by Tukey's post-test. No significant association between IL-23A (rs11171806) and levels of IL-23 was observed (i). A p-value of less than 0.05 was taken as significant</figcaption></figure>

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 activ-
ity of IL-17 cells and the production of various proin-
flammatory mediators in the body [9, 31]. IL-17A is a
multifunctional cytokine which has a protective role
in immunity for the clearance of intracellular patho-
gens such as Plasmodium spp. [9–11, 19, 32, 33], mak-
ing 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 lev-
els. 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 gen-
otyped *IL-17A* variants in Senegalese severe and uncom-
plicated 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 dis-
eases [5, 19, 35, 36]. IL-17 production is associated with
a very high occurrence of chronic inflammation and
immunopathological conditions [31]. Recent data sug-
gest 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 sensi-
tive 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 interleu-
kin-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 varia-
tions of the IL-17A gene. We identified 8 SNPs in the IL-
17A gene; among them, 6 SNPs (rs9791323, rs3819024,
s2275913, rs3819025, rs17880588 and rs3748067) were
detected with high frequencies (with MAF > 3%) at oppo-
site to 2 other SNPs (rs8193037 and rs8193038) with
MAF < 3% were observed. Then, statistical IL-17A poly-
morphism analysis was performed using logistic regres-
sion 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 vari-
ants 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) polymor-
phisms independently modulate susceptibility to Cer-
ebral 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 scenar-
ios, which in turn influence the outcome of malaria dis-
ease 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].

## Conclusions

The current report revealed an elevated level of IL-17A in severe malaria patients compared to healthy controls in Senegalese cohort. Furthermore, heterozygous mutant and minor alleles of *IL-17A* rs3819024 and rs3748067 polymorphisms predisposed subjects for the development of SM. Interestingly, the current report further validated the functional relevance of *IL-17A* (rs8193038) variants and demonstrated the association of mutants with elevated IL-17A levels. However, further studies, including more significant sample sizes in the different populations, are required to validate the observations of the present study. In addition, further investigation on the role of IL-17 and its interplay with other immune factors needs to be conducted in clinical settings. In the future, we plan to carry out a mechanistic study to understand the role of these *IL-17A* SNPs in gene expression. We intend to measure the expression of IL-17A mRNA to confirm our results. We will use a genome-wide association study to screen the IL-17A gene better and characterize the position of the SNPs better. Finally, we will perform proteomic studies and use bioinformatics tools to understand the molecular function of SNPs.

## Abbreviations

COVID Coronavirus disease 2019

CTR Control

EDTA Ethylene Diamine Tetraacetic Acid

ELISA Enzyme-Linked Immuno Assay

IFN Interferon

IL Interleukin

MAF Minor Allele Frequency

PCR Polymerase Chain Reaction

SM Severe Malaria

SNP Single Nucleotide Polymorphism

TGF Tumor Growth Factor

TNF Tumor Necrosis Factor

UCAD Universite Cheikh Anta Diop

UM Uncomplicated Malaria

WHO World Health Organization

## Acknowledgements

The authors are grateful to all the patients and the medical staff who have generously collaborated in the Malaria Genomic Project Clayton Dedonder 2014 (Institut Pasteur de Dakar). In addition, the authors thank the team of the Centre National de Recherche en Génétique humaine (CNRGH) for assistance in performing the genotyping. The French Ministry of Education and Research supported the CNRGH-CEA.

## Authors' contributions

FT conceived the study and the methodology and drafted the manuscript. GD conceived the study and the methodology. JFZ and CC conducted the data analysis, revised the manuscript and approved the final version. CD conducted the methodology, performed Sanger Sequencing and molecular biology experiments and approved the final version. BM conducted the malaria cohort recruitment, revised the manuscript from the hospital center, and approved the final version. AAMD, AT, MD, CMN, YD contributed to the correction and revision of the manuscript and approved the final version. JFD & AD coordinated this study, revised the manuscript, and approved the final version. All authors read and approved the final manuscript.

## Funding

This research received no external funding.

## Availability of data and materials

The data and materials supporting the conclusions of the study are available from the corresponding author on request.

## Declarations

### Ethics approval and consent to participate

"The study was conducted in accordance with the Declaration of Helsinki, and approved by the Ethics Committee of UCAD's Committee on Research and Ethics (CER) considers that the research proposed respects the appropriate ethical standard and, as a result, approves its execution under "Protocole 0344/2018/CER-UCAD". Written informed consent was obtained from adult participants and parents or legal representatives of children and also was obtained from the patient(s) to publish this paper.

### Consent for publication

Not applicable.

### Competing interests

The authors declare no competing interests.

### Author details

¹Groupe de Recherche Biotechnologies Appliquees & Bioprocedes Environnementaux, Ecole Superieure Polytechnique, Universite Cheikh Anta Diop de Dakar, Corniche Ouest, Dakar-Fann BP: 5085, Senegal. ²Departement de Biologie Animale, Faculte Des Sciences Et Techniques, Unite Postulante de Biologie GenetiqueGenomique Et Bio-Informatique (G2B), Universite Cheikh Anta DIOP, Avenue Cheikh Anta DIOP, Dakar BP: 5005, Senegal. ³Pole d'Immunophysiopathologie & Maladies Infectieuses (IMI), Institut Pasteur de Dakar, 36, Avenue Pasteur, Dakar BP: 220, Senegal. ⁴Equipe GBA «Genomique-Bioinformatique & Applications», Conservatoire National Des Arts Et Metiers, 292, Rue Saint Martin, Paris Cedex 03, Paris 75141, France. ⁵Centre National de Recherche en Génétique Humaine (CNRGH), Institut de Biologie François Jacob, 2 Rue Gaston Crémieux, CP 5721, Evry Cedex 91057, France. ⁶Pôle de Microbiologie, Institut Pasteur de Dakar, 36 Avenue Pasteur, Dakar BP 220, Senegal. ⁷Service d'Immunologie, Faculté de Médecine, de Pharmacie Et
d'Odontostomatologie, Université Cheikh Anta DIOP, Avenue Cheikh Anta DIOP, Dakar BP: 5005, Senegal.

Received: 16 June 2023 Accepted: 17 February 2024

Published online: 04 March 2024

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