|Year : 2020 | Volume
| Issue : 1 | Page : 23-30
Role of IL-17 gene polymorphism in Indian kala-azar
R Khatonier1, AM Khan1, P Sarmah1, GU Ahmed2
1 ICMR–Regional Medical Research Centre, Northeastern Region, Dibrugarh, India
2 Department of Biotechnology, Gauhati University, Guwahati, India
|Date of Submission||14-Mar-2018|
|Date of Acceptance||15-Nov-2018|
|Date of Web Publication||05-Feb-2021|
Dr A M Khan
ICMR–Regional Medical Research Centre, Division of Entomology and Filariasis, Northeastern Region, Post Box No. 105, Dibrugarh–786 001, Assam
Source of Support: None, Conflict of Interest: None
Background & objectives: Visceral leishmaniasis or kala-azar is a fatal protozoan disease caused by an obligate intracellular parasite, Leishmania donovani. Susceptibility, establishment of infection and severity of this disease depend upon many factors, but it is the host immune system that plays decisive role in disease progression. Keeping this view into consideration, we investigated the probable relationship between polymorphisms rs2275913 and rs8193036 in IL-17 gene, and its association as a risk factor for kala-azar in an endemic population of Assam, India.
Methods: A total of 209 subjects, 76 kala-azar cases (male: 46, female: 30, mean age ± SD: 34.60 ± 12.61) and 133 controls (male: 66, female: 67, mean age ± SD: 33.35 ± 14.48) were included in this study. We analysed the polymorphisms, rs2275913 and rs8193036 by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. The data were analysed using logistic regression analysis and SPSS software.
Results: The results revealed that the mutant rs8193036 TT genotype conferred 4.7-fold higher risk for kala-azar (p = 0.00991, OR = 4.72, 95% CI = 1.330–16.911). A significant difference was found between the allele frequencies of rs8193036 (p = 0.029, OR = 1.64, 95% CI = 1.04–2.57) when comparisons were done using the genetic models of association. When stratification analysis was done on the basis of active and past cases we found that during active infection rs2275913 A allele was significantly associated with increased risk of kala-azar (p = 0.016, OR = 3.95, 95% CI = 1.21–12.87).
Interpretation & conclusion: The findings revealed that IL-17 genetic variant, rs8193036 is an independent risk factor for kala-azar infection and may contribute in pathogenesis of the disease. Further, rs2275913 polymorphism of IL-17 gene is associated with kala-azar during active infection.
Keywords: Gene polymorphism; IL-17; kala-azar; Leishmania donovani; visceral leishmaniasis
|How to cite this article:|
Khatonier R, Khan A M, Sarmah P, Ahmed G U. Role of IL-17 gene polymorphism in Indian kala-azar. J Vector Borne Dis 2020;57:23-30
|How to cite this URL:|
Khatonier R, Khan A M, Sarmah P, Ahmed G U. Role of IL-17 gene polymorphism in Indian kala-azar. J Vector Borne Dis [serial online] 2020 [cited 2021 Apr 17];57:23-30. Available from: https://www.jvbd.org/text.asp?2020/57/1/23/308795
| Introduction|| |
Visceral leishmaniasis (VL) is a vector-borne, protozoan infection caused by Leishmania donovani in India. The disease is prevalent worldwide with an estimated disease burden of 1.98 million disability adjusted life years and a death rate of 20,000 to 40,000 per annum. Most of the VL cases are reported from India, Bangladesh, Brazil, Sudan, South Sudan, and Ethiopia. Kala-azar is endemic in eastern states of India, namely Bihar, Jharkhand, Uttar Pradesh, West Bengal and some areas of Assam,.
The protozoan parasite modulates the host innate immune response into its own benefit that reflects the need for a highly efficient proinflammatory response for protection. Cytokines are the major orchestrators of the host immune response and they play a crucial role during host parasite interaction. Interleukin-17 (IL-17, also known as IL-17A) is a novel proinflammatory cytokine produced by a subset of CD4+ T-helper cells called Th17 cells,,,. It acts as a bridge between the innate and adaptive immune system and plays a crucial role in restricting the growth of parasites by recruiting neutrophils to the site of infection. It is mostly produced by Th17 cells. However, this cytokine is also produced by other cells of the immune system like CD8+T cells, γδ T cells, invariant natural killer T cells (iNKT cells). There is very little information available regarding the cellular sources of this cytokine and the immune pathways followed by it.
Genetic variants in the cytokine genes contribute exclusively towards susceptibility or resistance against a disease when such variants occur in the course of pathway associated with protection against infection. The IL-17A gene is located on chromosome 6p12 and is composed of three exons and two introns. Single-nucleotide polymorphism (SNP) rs2275913 also known as G-197A is an SNP located within a binding motif for the nuclear factor of activated T cells (NFAT), which is a critical regulator of the IL-17 promoter and, therefore, has an effect on the IL-17 gene regulation. Genetic studies have found that IL-17 197A allele is associated with higher production of IL-17 and is associated with higher affinity for NFAT. The SNP rs8193036 is located at position 692 from the starting site of mRNA and 737 from the start codon of IL17A. The function of genetic variant rs8193036 is still poorly understood but it might regulate the expression of IL-17 through differential binding of transcription factor. The association of this polymorphism has been widely studied in different diseases like rheumatoid arthritis, cancer, Hepatitis C, ulcerative colitis, tuberculosis, Chagas disease and psoriasis; however, its role in VL is yet to be ascertained.
Literature retrieval revealed only few studies on IL-17 and its association with kala-azar globally; however, one study carried out in Iran on IL-17 gene polymorphism (rs1974226) and susceptibility to VL infection could not correlate between polymorphism in IL-17 and its association with susceptibility to VL infection. Keeping in view the evidences of recent research done on this aspect in other protozoan and non-parasitic infections/diseases where course of host immune system adopts similar approach in disease progression, we attempted this study to investigate, if there is any association between IL-17 genetic variations (rs2275913 and rs8193036) and risk of kala-azar in an endemic population of Assam.
| Material & Methods|| |
Selection of SNP
The SNPs for IL-17 gene polymorphism were selected with a minimum allele frequency of >10% in a population as derived from MAF count in NCBI SNP database, as there was no data available for the SNPs in the study and Indian population. Both the SNPs rs2275913 and rs8193036 are located within the non-coding region and, therefore, may regulate gene transcription.
Sampling method and study design
The present study is a case control study and cases were identified from the study area and defined as new cases, old cases with past history of kala-azar and cases under treatment. Matching healthy controls were selected from the same endemic area. At a two-sided confidence level of 95% with 80% sampling power and assuming 50% exposure, with a case to control ratio of approximately 1 : 2, sample size was calculated by Fleiss statistical methods for rates and proportion of 3.6 and 3.8. Sample size, assuming a dropout of 10% samples, a total of 76 cases and 133 healthy controls were taken in our study.
The study was carried out in Kamrup Metro district of Assam, India where kala-azar is endemic. Active cases of kala-azar included those cases which were reported during 2008–2009 epidemic and also that appeared later till 2012. Samples collected during the case detection from December 2008–February 2009 were stored at –80°C in the laboratory. Past cases of kala-azar are those cases that were positive for kala-azar, during 2008–2012 and samples were collected in follow-up in the period 2014–2016. Both males and females above 5 yr of age were included in the study. The study included 76 kala-azar cases (46 males and 30 females) and 133 healthy controls (66 males and 67 females). Kala-azar was diagnosed using rk39 rapid diagnostic test and confirmed by PCR using primers specific for L. donovani. The controls were tested for kala-azar and were found negative by both rapid diagnostic test and PCR.
About 5 ml of venous blood was collected from the participants in EDTA coated vials and stored at –20°C until use. DNA was extracted from the blood samples using DNeasy Blood and tissue kit (Qiagen) according to the manufacturer’s instructions. The extracted DNA was stored at –20°C until use. The primer for rs8193036 SNP was designed in the study using the Primer Quest tool software from IDT. The restriction sites in the amplified region were determined by restriction mapper and NEB cutter. The SNP rs2275913 was analysed using primers from previously published literature. The PCR-RFLP was used to study the genetic polymorphism in the IL-17 gene using restriction enzyme Ear I for rs2275913 and MBO II for rs8193036 [Table 1]. PCR was performed in a total reaction volume of 10 μl using readymade 2× PCR Master Mix (Promega), forward primer and reverse primer (0.3 pmol/μl) and 200–250 ng/μl DNA. The cycling conditions for PCR include an initial denaturation at 95°C for 5 min followed by 40 cycles of denaturation at 95°C for 30 sec, annealing at 58°C for 1 min, extension at 72°C for 1 min and a final extension of 72°C for 5 min. The amplified products were subjected to restriction digestion using specific restriction enzymes in a total reaction volume of 10 μl in the supplied reaction buffer. The restriction digestion products were run in 3% agarose gel and stained with ethidium bromide. The presence of specific-digested bands was observed under Gel documentation system.
Data analysis was done using logistic regression analysis (http://ihg.gsf.de/cgi-bin/hw/hwal.pl). Genotype and allele frequencies were calculated on kala-azar subjects and controls by direct gene counting. The statistical analysis of the differences between groups was performed by Chisquared test using SPSS software version 16. Hardy-Weinberg equilibrium was determined by comparing the observed genotype frequencies with those of the expected. The linkage disequilibrium (LD) between the two polymorphisms was calculated using Haploview version 4.2 program. The p-value <0.05 was considered significant.
This study was approved by the ethics committee of ICMR–Regional Medical Research Centre (RMRC), Dibrugarh. Written informed consent was taken from all the participants included in this study.
| Results|| |
Demographic characteristics of the study population
To determine IL-17 genetic variants as a risk factor for kala-azar 133 healthy controls and 76 subjects with kala-azar from the same endemic area were genotyped using the PCR-RFLP method [Figure 1]a and [Figure 1]b. The percentage of males and females are shown in [Table 2]. There was no significant difference between the kala-azar subjects and healthy controls in terms of age and sex (p >0.05)
|Figure 1: Electrophoretogram of restriction enzyme digestion — (a) IL-17 SNP rs2275913; and (b) rs8193036. M–DNA ladder; G/G–GG genotype, A/G– AG genotype, T/T–TT genotype, C/C–CC genotype and C/T–CT genotype hp1. The results in this study, suggested that IL17A is a candidate gene that confers the genetic susceptibility for pediatric asthma in Taiwanese population. Furthermore, the results provided a genetic basis indicating that the expression regulation was involved in asthma pathological mechanism.|
Click here to view
Genotype distribution and allele frequencies of IL-17 genetic variants in subjects with kala-azar and controls
The genotypes distribution of IL-17 rs8193036 was in Hardy-Weinberg equilibrium among the controls while the distribution of IL-17 rs2275913 was not. The genotype and allele frequencies of the two polymorphisms in the IL-17 gene (rs8193036 and rs2275913) are demonstrated in [Table 3] and [Table 4].
|Table 3: Genotype and allele frequencies for the single nucleotide polymorphism rs8193036 in the subjects with kala-azar and controls|
Click here to view
|Table 4: Genotype and allele frequencies for the single nucleotide polymorphism rs2275913 in the subjects with kala-azar and controls|
Click here to view
For SNP IL-17 rs8193036, when genotype comparison was done using genetic models of association, individuals homozygous for rs8193036 T allele, had 4.7-fold risk for kala-azar than individuals homozygous for rs8193036C allele (p = 0.00991, OR= 4.72, 95% CI = 1.33–16.91) and this was statistically significant. When association analysis was done at allelic level, a significant difference was observed between the rs8193036 T allele and rs8193036 C allele (p = 0.029, OR = 1.64, 95% CI = 1.04–2.57). No significant association was found between the distribution of genotypes and alleles for rs2275913 among the cases and controls in any of the genetic models of association. We analysed genotype and allele frequencies of rs8193036 and rs2275913 in kala-azar subjects and controls after stratifying according to gender [Table 5] and found that in the female group the T allele of SNP rs8193036 was significantly associated with increased risk of kala-azar (p = 0.04, OR = 2.01, 95% CI = 0.99–4.09).
|Table 5: Genotype and allele frequencies of the rs2275913 and rs8193036 in the study population stratified by gender|
Click here to view
We further analysed the genotype and allele frequencies of rs8193036 and rs2275913 on the basis of active infection (n = 28) and past infection (n = 47). Statistical analysis of the subjects under treatment could not be performed due to only one case and, therefore, was excluded from the analysis. For SNP rs8193036, we could not find any significant association among the genotypes and alleles, both in the active and past cases and also after analysing using genetic models of association. Interestingly, we found that among the subjects with active infection, the rs2275913 A allele was significantly associated with increased risk of kala-azar (p = 0.016, OR = 3.95, 95% CI = 1.21–12.87). Similarly, among the subjects with past infection the rs2275913 A allele was marginally associated with kala-azar (p = 0.047, OR = 0.54, 95% CI = 0.29–0.99) [Table 6].
|Table 6: Genotype and allele frequencies of rs2275913 and rs8193036, stratified according to active and past infection|
Click here to view
The LD between the two polymorphisms was calculated wherein, the r2 and D’ values of the LD were 0.028 and 0.422, respectively with a confidence bounds of 0.09 to 0.68. Therefore, it suggests that the two SNPs are in linkage equilibrium and independently associated with kala-azar.
| Discussion|| |
In the present study, we tried to investigate any plausible relationship between IL-17 genetic variants with risk of kala-azar in a population endemic for kala-azar. Analysis of results revealed that individuals carrying the homozygous recessive rs8193036 TT genotype and T allele possess higher risk for kala-azar. When stratification analysis was done according to gender, a significant difference in cases (21.66%) compared to controls (35.2%) was observed between the rs8193036 T allele in the females which suggests that the females carrying the mutant allele are more prone to kala-azar than males. In our study, we could not find significant difference between allele and genotype frequencies of the SNP rs2275913. However, when stratification analysis was done on the basis of active and past infection we found that during active infection individuals carrying the rs2275913 A allele had higher risk for kala-azar which suggests that the mutant A allele might contribute to genetic predisposition of kala-azar.
The importance of IL-17 in context of leishmaniasis was first described by Pitta et al in 2009 in a study conducted in Sudan. Their study suggests that L. donovani produces the cytokines IL-17 and IL-22, which play complementary roles in protection against infection. Thus, the findings of our study may suggest that the SNPs investigated may affect IL-17 level; however, association of IL-17 SNP with IL-17 protein level was not done in this study. A genetic association study of IL-17 genetic variant rs1974226 conducted in kala-azar in an Iranian population suggests that there is no association between IL-17 SNP rs1974226 and susceptibility to kala-azar. In contrast to that study, the findings of our study suggests a positive association of IL-17 genetic variant with kala-azar, however, this might be because the SNPs investigated in this study were different. Nevertheless, there is dearth of information regarding genetic polymorphisms of IL-17 gene in context of leishmaniasis.
There is published literature which suggests that IL-17 genetic variant is a crucial factor that contributes to genetic predisposition to many diseases like cancers, viral diseases, bacterial diseases, autoimmune diseases, etc. The relevance of the studied SNPs in different diseases involving different populations has been made in several studies. A study done by Shibata et al found that rs2275913 of IL-17A was significantly associated with the development of gastric cancer which was concordant with the findings of Qinghai et al wherein they reported an association of the polymorphism with increased risk of gastric cancer in a Chinese population. Similar to our findings, genetic association of rs8193036 was also reported in inflammatory diseases like asthma in a Taiwanese population. The influence of IL-17 genetic variants on IL-17 production was revealed by two studies done by Chen et al and Espinoza et al. Espinoza et al suggested that individuals carrying AA/AG genotype secreted more IL-17 than individuals possessing GG genotype. However, Chen et al did not find any significant difference in IL-17 levels among the different genotypes. Another study in inflammatory bowel disease has correlated the association of the IL-17 genetic variant with IL-17 mRNA expression and they found that rs8193036 T allele was associated with higher IL-17 mRNA levels. Similarly, in tuberculosis infection rs2275913 A allele was associated with protection against TB and that individuals carrying the A allele produced more IL-17. In contrast, Rasouli et al could not find any association between the SNPs rs2275913 and rs8193036 SNP with brucellosis. The role of these SNPs have been implicated in case of viral diseases wherein SNP rs2275913 was found in association with genetic predisposition to chronic HBV infection and risk of hepatocellular carcinoma, and that the rs2275913 GG genotype produce more IL-17 while rs8193036 contribute to resistance against HBV infection. Literature search could not reveal similar studies done elsewhere on IL-17 SNP rs2275913 and rs8193036 in association with kala-azar to correlate the findings of the present study. Further, due to the sampling nature of the study, the effect of these SNPs in the IL-17 level was not attempted. This is one of the limitations of the present study.
| Conclusion|| |
In conclusion, the present study revealed that IL-17 genetic variant rs8193036 is associated with susceptibility to kala-azar and that individuals carrying the rs8193036TT genotype and T allele are more susceptible to kala-azar. Also, mutant A allele of IL-17 SNP rs2275913 could be considered as susceptibility factor of kala-azar. To our knowledge this is the first study on IL-17 SNP rs2275913 and rs8193036 and is thought to provide novel genetic information regarding the knowledge on IL-17 genetic variants in disease predisposition. Further studies on the effect of genetic polymorphisms on IL-17 levels, at both the mRNA and protein level, would strengthen the results of this study.
Conflict of interest
The authors have no conflict of interest.
| Acknowledgements|| |
The authors gratefully acknowledge the Medical Officer and supporting health personnel of the Mini PHC (Panikheti) of Kamrup Metro district, Assam, India for their assistance in the collection of biological samples and field work during the study. RK thanks Department of Science and Technology (DST), New Delhi for financial support in the form of DST-INSPIRE fellowship to carry out this study. The authors also thank the Director, ICMR–RMRC, Dibrugarh for providing laboratory facilities to conduct the present study.
| References|| |
Alvar J, Vélez ID, Bern C, Herrero M, Desjeux P, Cano J, et al
. WHO leishmaniasis control team. Leishmaniasis worldwide and global estimates of its incidence. PLoS One
van Griensven J, Zijlstra EE, Hailu A. Visceral leishmaniasis and HIV coinfection: Time for concerted action. PLoS Negl Trop Dis
Verma SK, Ahmad S, Shirazi N, Kusum A, Kaushik RM, Barthwal SP. Sodium stibogluconate-sensitive visceral leishmaniasis in the non-endemic hilly region of Uttarakhand, India. Trans R Soc Trop Med Hyg
Liu D, Uzonna JE. The early interaction of Leishmania with macrophages and dendritic cells and its influence on the host immune response. Front Cell Infect Microbiol
Harrington LE, Hatton RD, Mangan PR, Turner H, Murphy TL, Murphy KM, et al
. Interleukin 17-producing CD4+
effector T cells develop via
a lineage distinct from the T helper type 1 and 2 lineages. Nat Immunol
Langrish CL, Chen Y, Blumenschein WM, Mattson J, Basham B, Sedgwick JD, et al
. IL-23 drives a pathogenic T cell population that induces autoimmune inflammation. J Exp Med
Park H, Li Z, Yang XO, Chang SH, Nurieva R, Wang YH, et al
. A distinct lineage of CD4 T cells regulates tissue inflammation by producing interleukin 17. Nat Immunol
Boniface K, Blumenschein WM, Brovont-Porth K, McGeachy MJ, Basham B, Desai B, et al
. Human Th17 cells comprise heterogeneous subsets including IFN-γ-producing cells with distinct properties from the Th1 lineage. J Immunol
Takatori H, Kanno Y, Watford WT, Tato CM, Weiss G, Ivanov II, et al
. Lymphoid tissue inducer-like cells are an innate source of IL-17 and IL-22. J Exp Med
Sheel M, Beattie L, Frame TC, de Labastida Rivera F, Faleiro RJ, Bunn PT, et al
. IL-17A-producing γδ T cells suppress early control of parasite growth by monocytes in the liver. J Immunol
Sadighi S, Bazmany A, Babaloo Z, Amadi H. Evaluation of interleukin 17: A polymorphism in patients with visceral leishmaniasis. J Anal Res Clin Med
Espinoza JL, Takami A, Nakata K, Onizuka M, Kawase T, Akiyama H, et al
. A genetic variant in the IL-17 promoter is functionally associated with acute graft-versus-host disease after unrelated bone marrow transplantation. PLoS One
Arisawa T, Tahara T, Shibata T, Nagasaka M, Nakamura M, Kamiya Y, et al
. The influence of polymorphisms of interleukin-17A
genes on the susceptibility to ulcerative colitis. J Clin Immunol
Aouizerat BE, Dhruva A, Paul SM, Cooper BA, Kober KM, Miaskowski C. Phenotypic and molecular evidence suggests that decrements in morning and evening energy are distinct but related symptoms. J Pain Symp Manag
Nordang GB, Viken MK, Hollis-Moffatt JE, Merriman TR, Førre ØT, Helgetveit K, et al
. Association analysis of the interleukin 17A
gene in Caucasian rheumatoid arthritis patients from Norway and New Zealand. Rheumatology (Oxford)
Dai ZM, Zhang TS, Lin S, Zhang WG, Liu J, Cao XM, et al
. Role of IL-17A rs2275913 and IL-17F rs763780 polymorphisms in risk of cancer development: An updated meta-analysis. Sci Rep
Nan YM, Zhang YG, Zheng HW, An CM, Li YS, Zhang Y, et al
. Individualized treatment strategies and predictors of virological response for chronic hepatitis C: A multicenter prospective study from China. Int J Clin Exp Med
Hayashi R, Tahara T, Shiroeda H, Saito T, Nakamura M, Tsutsumi M, et al
. Influence of IL17A polymorphisms (rs2275913 and rs3748067) on the susceptibility to ulcerative colitis. Clin Exp Med
Milano M, Moraes MO, Rodenbusch R, Carvalho CX, Delcroix M, Mousquer G, et al
. Single nucleotide polymorphisms in IL17A and IL6 are associated with decreased risk for pulmonary tuberculosis in Southern Brazilian population. PLoS One
Leon Rodriguez DA, Echeverría LE, González CI, Martin J. Investigation of the role of IL17A
gene variants in Chagas disease. Genes Immun
Prieto-Pérez R, Solano-López G, Cabaleiro T, Román M, Ochoa D, Talegón M, et al
. The polymorphism rs763780 in the IL-17F gene is associated with response to biological drugs in patients with psoriasis. Pharmacogenomics
Khan AM, Pandey K, Kumar V, Dutta P, Das P, Mahanta J. Sample survey for indigenous cases of kala-azar in Assam by rk39 dipstick test. Indian J Med Res
Khan AM, Dutta P, Khan SA, Baruah SK, Raja D, Khound K. Kala-azar and post-kala-azar dermal leishmaniasis, Assam, India. Emerg Infect Dis
Rasouli M, Asaei S, Kalani M, Kiany S, Moravej A. Interleukin-17A genetic variants can confer resistance to brucellosis in Iranian population. Cytokine
Pitta MG, Romano A, Cabantous S, Henri S, Hammad A, Kouriba B, et al
. IL-17 and IL-22 are associated with protection against human kala-azar caused by Leishmania donovani. J Clin Invest
Shibata T, Tahara T, Hirata I, Arisawa T. Genetic polymorphism of interleukin-17A and-17F genes in gastric carcinogenesis. Human Immunol
Qinghai Z, Yanying W, Yunfang C, Xukui Z, Xiaoqiao Z. Effect of interleukin- 17A
and interleukin-17F gene polymorphisms on the risk of gastric cancer in a Chinese population. Gene
Wang JY, Shyur SD, Wang WH, Liou YH, Lin CG, Wu YJ, et al
. The polymorphisms of interleukin 17A (IL17A)
gene and its association with pediatric asthma in Taiwanese population. Allergy
Chen J, Deng Y, Zhao J, Luo Z, Peng W, Yang J, et al
. The polymorphism of IL-17 G-152A was associated with childhood asthma and bacterial colonization of the hypopharynx in bronchiolitis. J Clin Immunol
Kim SW, Kim ES, Moon CM, Park JJ, Kim TI, Kim WH, et al
. Genetic polymorphisms of IL-23R and IL-17A and novel insights into their associations with inflammatory bowel disease. Gut
Rolandelli A, Del Pino RH, Pellegrini JM, Tateosian NL, Amiano NO, de La Barrera S, et al
. The IL-17A rs2275913 single nucleotide polymorphism is associated with protection to tuberculosis but related to higher disease severity in Argentina. Sci Rep
Li N, Zhu Q, Li Z, Han Q, Zhang G, Chen J, et al. IL17A
gene polymorphisms, serum IL17A and IgE levels, and hepatocellular carcinoma risk in patients with chronic hepatitis B virus infection. Mol Carcinog
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]