rs2243268 and rs2243274 of interleukin-4 (il-4) gene are associated with reduced risk for...

Infection, Genetics and Evolution 23 (2014) 121–128

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Infection, Genetics and Evolution

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rs2243268 and rs2243274 of Interleukin-4 (IL-4) gene are associatedwith reduced risk for extrapulmonary and severe tuberculosis in ChineseHan children

http://dx.doi.org/10.1016/j.meegid.2014.01.0311567-1348/� 2014 Elsevier B.V. All rights reserved.

Abbreviations: TB, tuberculosis; SNP, single-nucleotide polymorphism; EPTB,extrapulmonary tuberculosis; IL-4, interleukin-4.⇑ Corresponding author. Tel.: +86 010 59616983; fax: +86 010 59718662.

E-mail address: [email protected] (A-Dong Shen).

Hui Qi a, Lin Sun a, Ya-Qiong Jin a, Chen Shen a, Ping Chu a, Sheng-Feng Wang b, Qing-Qin Yin a, Zhan Qi a,Fang Xu a, Wei-Wei Jiao a, Xi-Rong Wu a, Jian-Ling Tian a, Jing Xiao a, A-Dong Shen a,⇑a Key Laboratory of Major Diseases in Children and National Key Discipline of Pediatrics (Capital Medical University), Ministry of Education, Beijing Pediatric Research Institute,Beijing Children’s Hospital, Capital Medical University, No. 56, Nan-li-shi Road, Beijing 100045, Chinab Department of Epidemiology and Bio-statistics, School of Public Health, Peking University Health Science Center, Beijing 100191, China

a r t i c l e i n f o

Article history:Received 11 November 2013Received in revised form 17 January 2014Accepted 23 January 2014Available online 8 February 2014

Keywords:Interleukin-4Interleukin-10PolymorphismTuberculosisExtrapulmonary tuberculosisSevere tuberculosis

a b s t r a c t

Interleukin-4 (IL-4) and IL-10, which are produced by Th2 cells, serve as anti-inflammatory cytokines inthe immune responses to tuberculosis (TB). In order to investigate the association between susceptibilityto TB and single-nucleotide polymorphisms (SNPs) of the IL-4 and IL-10 genes, a case–control studyincluding 346 TB patients and 374 healthy controls was performed in Chinese Han children in NorthChina. Though no significant differences in the allelic and genotypic distributions of SNPs of these twogenes were observed between control group and TB group, rs2243268-A and rs2243274-G of the IL-4gene were associated with reduced risk of developing extrapulmonary tuberculosis (EPTB)(Prs2243268 = 0.005 and Prs2243274 = 0.004) and severe TB (Prs2243268 = 0.003 and Prs2243274 = 0.003). Thehaplotype comprising rs2243268-A and rs2243274-G was found to be a resistance factor against EPTBand severe TB. In addition, after stimulation with inactivated H37Rv, blood samples of the rs2243268AA + AC carriers showed significantly reduced IL-10 production (P = 0.045) compared to the CC carriers.In conclusion, rs2243268-A and rs2243274-G of the IL-4 gene were found to confer resistance to EPTB andsevere TB in Chinese Han children.

� 2014 Elsevier B.V. All rights reserved.

1. Introduction

Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB)is a re-emerging disease in many parts of the world. TBthreatens human health and places a heavy financial burdenon the family and society. In the 2013 Global TuberculosisReport, the World Health Organization (WHO) indicated thatthere were an estimated 8.6 million new cases of TB and 1.3million deaths from TB in 2012 (World Health Organization,2013). More than one third of the global population is infectedwith MTB, but only 10% develop clinical TB (Arend et al.,2001). TB development is affected by many factors, includingstrain virulence, environmental factors, and various risk factors,including malnutrition, human immunodeficiency virus (HIV)

infection, and immunosuppressive therapy (Mathema et al.,2006). However, only a minority of patients with TB haveidentifiable risk factors, suggesting that host genetic factorsinfluence MTB infection outcomes.

Immune responses to TB, including innate and adaptiveimmune responses, are regulated by interactions between immunecells and the cytokines secreted by these cells (Selvaraj et al.,2008). Macrophages and CD4+ T lymphocytes, which include Thelper (Th) 1 cells, Th2 cells, and Th17 cells, play important rolein innate and adaptive immune responses. After phagocytosis,MTB is processed within macrophages and dendritic cells andpresented to CD4+ T cells in regional lymph nodes on majorhistocompatibility complex (MHC) class II molecules. Interleukin-12 (IL-12) is secreted by macrophages. It can cause Th1 cells toproliferate and produce interferon (IFN)-c. IFN-c, produced byCD4+ T cells (mostly Th1), CD8+ T cells and natural killer (NK) cells,activates the macrophages, causing them to become microbicidal.Th17 cells modulate the immune response to MTB by producingIL-17A (Basu et al., 2012).

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122 H. Qi et al. / Infection, Genetics and Evolution 23 (2014) 121–128

IL-4 and IL-10, which are produced by Th2 cells, play importantroles in infectious diseases (Biedermann et al., 2001). IL-4 and IL-10act as anti-inflammatory cytokines in the immune responses thattake place in case of TB infection (Hussain et al., 2002). They havebeen shown to downregulate IFN-c, and thus have a deleteriouseffect on TB patients. Levels of IL-4 and IL-10 are elevated duringthe advanced stages of TB, and they downregulate Th1 responses(Wangoo et al., 2001). Although IL-4 and IL10 downregulate IFN-c responses, they are necessary to maintain a balance for appropri-ate anti-MTB activity (Higgins et al., 2009).

The association between single-nucleotide polymorphisms(SNPs) of IL-4 gene and susceptibility to TB has been studied inIranians and Indians, and the similar studies of SNPs in IL-10 geneand susceptibility to TB have been conducted in Egyptians andIndians. In Iranians, IL-4 -590 (rs2243250) C allele and the T/Cgenotypes were found to be significantly more common in TBpatients than in controls, and the -1098 (rs2243248) and -33(rs2070874) were not found to be associated with susceptibilityto TB (Amirzargar et al., 2006). A statistically significant differencein the frequency of GG genotype of -1082 (rs1800896) SNP of IL-10gene was found between patients with PTB and extrapulmonary TB(EPTB) in Egyptians (Mosaad et al., 2010). In Indians, the higher fre-quency of CT genotype rs2243250 SNP of IL-4 in pulmonary TB(PTB) was found in South, suggesting an association betweenrs2243250 and the susceptibility to TB and protective role of theCC genotype (Vidyarani et al., 2006). However, a study in North In-dia did not find any association between rs2070874 SNP of IL-4gene and TB (Abhimanyu et al., 2012). In addition, rs3024498SNP of IL-10 gene did not show any strong relevance to immunityto TB in Indians (Abhimanyu et al., 2011).

Pediatric TB is an important part of the burden that TB places onsociety. Children are much more likely than adults to develop TBdisease after exposure, and this development is both more rapidand more likely to become extrapulmonary TB (EPTB), dissemi-nated TB (DTB), or tuberculous meningitis (TBM) (Walls and Shing-adia, 2004; Wu et al., 2012). It has been suggested that DTB mayreflect Mendelian predispositions in a fraction of children (3–30%estimated by Bayesian statistics), and adult pulmonary TB (PTB)may involve more complex genetic predispositions (Alcais et al.,2005; Casanova and Abel, 2007). Here, we present results of thefirst study of the IL-4 and IL-10 SNPs and their contribution to sus-ceptibility to clinical TB in a BCG-vaccinated pediatric populationin North China.

2. Materials and methods

2.1. Ethics statement

The clinical investigation Rs2243268 and Rs2243274 ofInterleukin-4 (IL-4) Gene were Associated with Reduced Risk forDeveloping Extrapulmonary Tuberculosis and Severe Tuberculosisin Chinese Han Children has been conducted according to theprinciples expressed in the Declaration of Helsinki. For researchinvolving human participants, informed consent has been obtainedfrom the patients or the guardian of the patients. The research hasbeen approved by the Ethics Committee of the Beijing Children’sHospital.

2.2. Study sample

The data from 346 case patients and 374 control subjects wereincluded in this study. All study individuals were of the Han Chi-nese ethnicity. All the cases and controls were vaccinated withBCG at birth, which was confirmed by the presence of a scar inthe left shoulder and vaccination records in the clinical charts. A

total of 346 blood samples were obtained from pediatric patientswith TB admitted at the Beijing Children’s Hospital (affiliated withCapital Medical University; Beijing, China) between February 2005and October 2011. The pediatric TB patients originated from Bei-jing and surrounding provinces in northern China. Patients werediagnosed to be PTB (a patient with TB disease involving the lungparenchyma), EPTB (TB affecting extrapulmonary sites within thebody exclusively or in combination with PTB), severe TB (patientswith disseminated TB (DTB) or TBM; DTB refers to TB with multi-organ involvement and military radiographic pattern) or other TB(other than severe TB) by at least two experienced pediatriciansin Beijing Children’s Hospital according to the pediatric TB clinicaldiagnosis standard (Cuevas et al., 2012; Graham et al., 2012; Norbiset al., 2013; Wu et al., 2012). The diagnosis of pediatric TB is basedon a the following factors: (1) clinical presentation (symptoms orsigns); (2) imaging (chest radiography and computed tomographyscan); (3) contact history (family and close contact); (4) purifiedprotein derivative (PPD) tuberculin skin test (using 5 TU); (5) posi-tive clinical response to anti-TB therapy; (6) except other lung dis-ease, such as the various reasons pneumonia, lung tumor and soon. Clinical TB could be diagnosed if positive features of (1) plus(2) or (1) plus either two of (3)–(6) were present (SupplementaryTable 1).

Control group subjects were recruited from those admitted tothe Beijing Children’s Hospital between June 2005 and October2011(Feng et al., 2011). Though children in Beijing Children’s Hos-pital were from all over China, we selected Han Chinese Children inNorth China as our studied samples to eliminate the differences ofgenetic background. The samples were mostly from Beijing, Hebei,Henan, Shanxi and Inner Mongolia in North China; the percentageof children in these four provinces in control group was 81.0% andthe percentage in case group was 72.5%. Controls were matchedwith TB patients by age, gender and ethnicity. The control groupincluded 374 children admitted at the hospital for physical exam-ination without history of TB with normal radiographic examina-tion findings, and with PPD skin test results <5 mm. The controlgroup children were without the infectious diseases or the historyof any inflammatory or autoimmune diseases, including TB andHIV infection. However, as we are dealing with a pediatric group,the possibility of a control to evolve as a case in future cannot beruled out. All controls were therefore seen or questioned again2 years after their initial visit, and their non-infection status wasconfirmed (with PPD skin test results <5 mm).

Blood samples from children with TB and controls were col-lected and stored at �80 �C. Genomic DNA was extracted from1 mL of EDTA anti-coagulated peripheral blood using the QIAampDNA Blood Midi Kit (Qiagen, Germany).

2.3. Selection of SNPs and genotyping analysis

We aimed to identify SNPs of the IL-4 gene reportedly associ-ated with TB susceptibility in other populations. The associationbetween rs2243250 and susceptibility to TB was studied in Iranianand Indian (Amirzargar et al., 2006; Vidyarani et al., 2006). As a re-sult, we have chosen rs2243250 and SNPs in linkage disequilibrium(LD) with rs2243250 for our research. According to CHB data of thepublic HapMap database (http://hapmap.ncbi.nlm.nih.gov), minorallele frequencies (MAFs) of these four SNPs in Chinese Han popu-lation were greater than 0.05. Haploview software 4.2 (Barrettet al., 2005; Barrett, 2009) was used to calculate LD of thesers2243250, rs2243282, rs2243268 and rs2243274 and strong LDwas detected in these four SNPs (r2 > 0.8). We selectedrs2243250 (promoter), rs2243268 (intron), rs2243274 (intron)and rs2243282 (intron) for our genotyping analysis. MassArray(Sequenom, USA) was used for genotyping all SNPs (Gabriel

http://hapmap.ncbi.nlm.nih.gov

Table 1Demographic characteristics of study population.

Characteristic Control (n = 374) All TB (n = 346) P

Gender 0.385b

Male (%) 222 (59.4) 217 (62.7)Female (%) 152 (40.6) 129 (37.3)

AgeMean (SD) 6.2 (3.8) 5.7 (4.7) 0.105c

TB typePTB 154 (44.5)EPTB 192 (55.5)

Severity of TBa

Other 189 (54.6)Severe 157 (45.4)

TB, tuberculosis; PTB, pulmonary tuberculosis; EPTB, extrapulmonary TB.a Severe TB: DTB and TBM; other TB: other than severe TB.b P value was calculated by chi-square tests.c P value was calculated by t test.

H. Qi et al. / Infection, Genetics and Evolution 23 (2014) 121–128 123

et al., 2009) and this assay was accomplished by Bio Miao Biolog-ical Technology (Beijing, China).

As MAF of two reported SNPs (rs1800896 and rs3024498) ofIL-10 gene in Chinese Han population was lower than 0.05, we havechosen tag SNPs of IL-10 gene for our genotyping. The tag SNPwas selected based on ability to tag surrounding variants (Chr1:205,002,571–205,017,462) in the Han Chinese panel (Beijing,China) of the International HapMap project, NCBI build B36 assem-bly HapMap phase II (http://www.hapmap.org). Tagger softwareincluded in Haploview software 4.2 was also used to select tagSNP. The region used in tag SNP selection included a region from5000 bp 50upstream to 5000 bp 30downstream of IL-10.Rs1800871 (promoter), rs3021094 (intron) and rs3790622 (intron)were the tag SNPs in IL-10 gene obtained by aggressive taggingMAF of 0.05 and pairwise correlation coefficient r2 of 0.80. LDwas also calculated and graphically displayed with the Haploview4.2 software.

2.4. Cytokine assays

Whole blood was harvested from children in control group withdifferent genotypes of rs2243268. There were 6 children of AAgenotype, 37 children of AC genotype and 25 children of CC geno-type. The cytokine assays were performed as described (Hawnet al., 2006). In brief, blood samples were prepared by dilutingvenous blood 1:5 with RPMI 1640 (Invitrogen, USA), plating in a96-well dish, stimulating by inactivated MTB H37Rv (the concen-tration of bacteria is 7.5 � 103 lL) for 20 h, and then harvestingsupernatants. The inactivated MTB H37Rv was kindly providedby Professor Kanglin Wan in National Institute for CommunicableDisease Control and Prevention, Chinese Center for Disease Controland Prevention. The inactivated H37Rv was obtained by heatkilling at 80 �C for 30 min (Doig et al., 2002).

The concentration of cytokines (IL-4, IL-10 and IL-17A) in thecell culture supernatant samples were quantified using a custom-ized Milliplex MAP Human Cytokine/Chemokine Panel (#HCYTO-MAG-60K, Millipore, USA).The assay was performed according tothe manufacturer’s instructions. Standards and samples wereanalyzed in duplicates on a Luminex 200 device (Luminex, USA)using the MilliplexTM Analyst Software (Version 3.5, Millipore,USA) (Moller et al., 2012).

2.5. Statistical analysis

Statistical analysis was carried out using the Statistical Packagefor SNPStats software (http://bioinfo.iconcologia.net/snpstats/start.htm) and SPSS 18.0 (Chicago, USA). The homogeneity analysiswas calculated by RevMan 4.2.8 (Oxford, UK). Unpaired t-tests (forcontinuous variables) and chi-square tests (for unordered categor-ical variable) were used to analyze differences among the groups.The Hardy–Weinberg equilibrium (HWE) was calculated usingchi-square goodness of fit. Adjusted odds ratio (AOR) and 95% con-fidence interval (CI) were calculated by logistic regression analysis.Stratification analysis was further performed by type and severityto evaluate the homogeneity within different variable-related ORfor risk of TB among three different genotypes of SNPs. Logisticregression (adjusting for gender and age) was used to analyze dif-ferences of genotype and allele frequencies of SNP polymorphismsin control group vs. TB group and control vs. TB subgroup. A P valueof less than 0.05 was considered nominally significant for homoge-neity analysis. If the P value was significant, Bonferroni correctionfor multiple testing of 7 SNPs (since 7 of the 8 targeted SNPs werefound polymorphic in our study) was additionally applied to thesingle-point results. Because rs2243268 and rs2243274 are in aclose block (r2 > 0.9) and rs1800871, rs3021094 and rs3790622are also in a close block (r2 > 0.9), an application of the Bonferroni

correction for multiple testing (4 markers/tests) reset the signifi-cance threshold from 0.05 to 0.0125.

3. Results

3.1. TB patients and uninfected controls

Demographic data for 346 case patients and 374 control sub-jects are included in Table 1. All study subjects were vaccinatedfor BCG at birth. The mean age in the TB patient group was5.7 years (SD = 4.7). The mean age in the control group (n = 374)was 6.2 years (SD = 3.8). There were no significant differencesbetween the groups with respect to age or gender.

Clinical TB types of 346 patients are listed in Table 1. The casegroup included 154 (44.5%) cases of active PTB and 192 (55.5%)cases of EPTB. Severe TB (TBM or DTB) was identified in 157(45.4%) patients. Other TB group (other than severe TB) included189 (54.6%) cases.

3.2. Analysis of HWE and the associations between SNPs of the IL-4and IL-10 genes with pediatric TB in China

SNPs (rs2243250, rs2243268, rs2243274, and rs2243282) of theIL-4 gene and (rs1800871, rs3021094, and rs3790622) of the IL-10gene were all found to be in HWE (P > 0.05) both in the TB and con-trol groups (data not shown). The genotyping results of detectedSNPs are summarized in Table 2. All these SNPs had MAFs over0.05 in the present population. No statistically significant differ-ences in allelic and genotypic distributions of these 7 SNPs wereobserved between the TB and control group (Table 2).

After adjustment for gender and age, statistical differences inthe frequencies of rs2243268 and rs2243274 were observedbetween the control vs. EPTB and control vs. severe TB groups(Table 3). In the control group vs. EPTB group, under a dominantmodel, the rs2243268-A and rs2243274-G alleles were found toact as resistance factors to EPTB (P = 0.005 and 0.004, respectively).In the control group vs. severe TB group, under a dominant model,the rs2243268-A and rs2243274-G alleles were found to act asresistance factors to severe TB (P values of the two SNPs are equal,P = 0.003). No statistically significant difference in rs2243268 orrs2243274 was observed between control vs. PTB and control vs.other TB. Except for rs2243268 and rs2243274, no statistical differ-ence in rs2243250, rs2243282 and SNPs of the IL-10 gene was ob-served in allelic and genotypic distributions between control vs. TBsubgroups.

http://www.hapmap.org

http://bioinfo.iconcologia.net/snpstats/start.htm

http://bioinfo.iconcologia.net/snpstats/start.htm

Table 2Genotyping of SNPs in the IL-4 and IL-10 gene between TB and control group in theHan Chinese pediatric population.

Gene Control n = 374 n (%) TB n = 346, n (%) a OR (95% CI)SNP

IL4rs2243250

T/T 218 (58.3) 209 (60.4) 1.00 (reference)C/T 129 (34.5) 123 (35.6) 0.99 (0.72–1.53)C/C 27 (7.2) 14 (4.0) 0.52 (0.27–1.03)P value 0.166C/T + C/C 156 (41.7) 137 (40.0) 0.91 (0.67–1.22)T 565 (75.5) 541(78.2) 1.00 (reference)C 183 (24.5) 151(21.8) 0.86 (0.67–1.10)

rs2243268C/C 210 (56.2) 215 (62.1) 1.00 (reference)A/C 143 (38.2) 114 (33.0) 0.79 (0.58–1.08)A/A 21 (5.6) 17 (4.9) 0.79 (0.41–1.55)P value 0.299C/A + A/A 164 (43.8) 131 (37.9) 0.79 (0.59–1.06)C 563 (75.3) 544 (78.6) 1.00 (reference)A 185 (24.7) 148 (21.4) 0.83(0.65–1.06)

rs2243274A/A 209 (55.9) 215 (62.1) 1.00 (reference)G/A 145 (38.8) 114 (33.0) 0.77 (0.57–1.06)G/G 20 (5.3) 17 (4.9) 0.82 (0.42–1.61)P value 0.260G/A + GG 165 (44.1) 131 (37.9) 0.78 (0.58–1.05)A 563 (75.3) 544 (78.6) 1.00 (reference)G 185 (24.7) 148 (21.4) 1.21 (0.94–1.55)

rs2243282A/A 223 (59.6) 211 (60.9) 1.00 (reference)C/A 124 (33.2) 122 (35.3) 1.03 (0.75–1.41)C/C 27 (7.2) 13 (3.8) 0.54 (0.25–1.00)P value 0.130C/A + CC 151 (40.4) 135 (39.1) 0.94 (0.70–1.27)A 570 (76.2) 544 (78.6) 1.00 (reference)C 178 (23.8) 148 (21.4) 1.14 (0.90–1.47)IL10

rs1800871T/T 155 (41.4) 145 (41.9) 1.00 (reference)C/T 182 (48.7) 162 (46.8) 0.95 (0.70–1.30)C/C 37 (9.9) 39 (11.3) 1.13 (0.68–1.88)P value 0.793C/T + C/C 219 (58.6) 201 (58.1) 0.98 (0.65–1.18)T 492 (65.8) 452 (65.3) 1.00 (reference)C 256 (34.2) 240 (34.7) 1.02 (0.82–1.27)

rs3021094A/A 110 (29.4) 106 (30.6) 1.00 (reference)C/A 190 (50.8) 168 (48.6) 0.91 (0.65–1.27)C/C 74 (19.8) 72 (20.8) 1.01 (0.66–1.53)P value 0.792C/A + CC 264 (70.6) 240 (69.4) 0.93 (0.68–1.29)A 410 (54.8) 380 (54.9) 1.00 (reference)C 338 (45.2) 312 (45.1) 1.00 (0.82–1.24)

rs3790622C/C 303 (81.0) 278 (80.3) 1.00 (reference)T/C 67 (17.9) 65 (18.8) 1.03 (0.71–1.51)T/T 4 (1.1) 3 (0.9) 0.83 (0.18-3.76)P value 0.957T/C + T/T 71 (19.0) 68 (19.7) 1.02 (0.70–1.48)C 673 (90.0) 621 (89.7) 1.00 (reference)T 75 (10.0) 71 (10.3) 0.97 (0.69–1.37)

TB: tuberculosis group.ORs were adjusted for sex and age in control group vs. TB group in a logisticregression model.Significant differences were indicated by a P value <0.05.


3.3. LD and haplotype analysis of rs2243268 and rs2243274 in thecontrol and TB groups

LD plot of 4 SNPs in the IL-4 gene showed the frequencies ofrs2243268 and rs2243274 to be similar in Chinese Han children

(Fig. 1). In control vs. TB group, control vs. PTB, and control vs.other TB, no statistical difference in haplotype of rs2243268 andrs2243274 was observed (data not shown). In control group vs.EPTB group and control group vs. severe TB group, the haplotypecomprising rs2243268-C and rs2243274-A was found to act as asusceptibility factor for EPTB (P = 0.008 and P = 0.006, respec-tively), and the haplotype comprising rs2243268-A andrs2243274-G was shown to act as a resistance factor to EPTB andsevere TB (P = 0.008 and P = 0.006, respectively) (Table 4).

3.4. Association of the AA + AC genotypes of rs2243268 with decreasedIL-10 production ex vivo

As rs2243268-A was found associated with susceptibility toEPTB and severe TB, the capacity of children with AA and AC geno-types to mediate cytokine production was examined using anex vivo assay. It showed that inactivated MTB stimulated IL-4,IL-10 and IL-17A production in the carriers of certain genotypes.Samples from individuals with rs2243268 AA + AC genotypes hadsignificantly less IL-10 production than those from individualswith the wild-type CC genotype of rs2243268 when stimulatedwith inactivated H37Rv (P = 0.045) (Fig. 2A). After stimulation withinactivated H37Rv, samples from individuals with the AA + ACgenotypes of rs2243268 had more IL-17A production than thosewith the CC genotype (P = 0.685) (Fig. 2B). We could not preciselymeasure the level of IL-4 since its level was lower than minimumdetectable concentration (4.5 pg/mL).

4. Discussion

The purpose of the present case–control study was to identifygenetic factors that confer individual susceptibility to pediatrictuberculosis. In this view, two of the studied IL-4 gene polymor-phisms, rs2243268 and rs2243274, provided an interesting clue.Though no significant differences of allelic and genotypic distribu-tions of rs2243268 and rs2243274 were detected between the con-trol group and unstratified TB group, minor alleles of both theseSNPs were associated with reduced risk of developing EPTB and se-vere TB. The haplotype containing mutated alleles of rs2243268-Aand rs2243274-G was also found to be a resistance factor againstEPTB and severe TB. In addition, after stimulation with inactivatedH37Rv, blood samples from children with the AA + AC genotypes ofrs2243268 showed significantly less IL-10 production and more IL-17A production than those with the CC genotype.

Many pieces of evidence have indicated that IL-4 is associatedwith the pathological process of TB. In addition to Th1 cytokines,there is also an IL-4 response in human TB. This response has beendetected in European patients (Seah et al., 2000; van Crevel et al.,2000) and in Africa (Lienhardt et al., 2002). TB patients experienceseveral IL-4-dependent phenomena, including production of IgEantibody to MTB (Rook et al., 2004) and increased expression ofdendritic cell-specific intercellular adhesion molecule 3-grabbingnonintegrin (DC-SIGN) (Tailleux et al., 2003). Similarly, high levelsof expression of both IL-4 mRNA and T cells containing IL-4 weredetected in human PTB (Seah et al., 2000). There is substantial evi-dence that an IL-4 response in TB downregulates protective Th1 re-sponses. A study by Wangoo et al. demonstrated that a pre-existingTh2 response was sufficient to undermine the efficacy of the dom-inant Th1 response (Wangoo et al., 2001). IL-4 has been shown todownregulate inducible nitric oxide synthase (iNOS), Toll-likereceptor 2 (TLR2), and macrophage activation, and this may deter-mine the outcome of MTB infection (Gordon, 2003; Sabroe et al.,2003).

SNPs of the IL-4 gene were reported to be associated with adultTB in Iranian and Indian individuals (Amirzargar et al., 2006;

Table 3Genotyping of SNPs of the IL-4 gene and IL-10 gene in control vs. PTB, control vs. EPTB, control vs. other TB and control vs. severe TB in the Han Chinese pediatric population.

Gene SNP Characteristics Control group (n = 374) TB group (n = 346) a OR (95% CI)a P valuea P valueb

1 + 2 n (%) 0 n (%) 1 + 2 n (%) 0 n (%) 1 + 2 vs. 0

IL4rs2243250 TB type

PTB (154) 156 (41.7) 218 (58.3) 66 (42.9) 88 (57.1) 1.05 (0.72–1.54) 0.785 0.37EPTB (192) 156 (41.7) 218 (58.3) 71 (46.1) 121 (53.9) 0.79 (0.55–1.14) 0.209

Severity of TBOther (189) 156 (41.7) 218 (58.3) 78 (41.3) 111 (58.7) 0.99 (0.69–1.41) 0.959 0.39Severe (157) 156 (41.7) 218 (58.3) 59 (37.6) 98 (62.4) 0.79 (0.54–1.17) 0.241

rs2243268 TB typePTB (154) 164 (43.9) 210 (56.1) 72 (46.8) 82 (53.2) 1.12 (0.77–1.63) 0.563 0.03EPTB (192) 164 (43.9) 210 (56.1) 59 (30.7) 133 (69.2) 0.59 (0.41–0.85) 0.005






IL10rs1800871 TB type

PTB (154) 219 (58.6) 155 (41.4) 89 (57.8) 65 (42.2) 0.97 (0.67–1.43) 0.891 0.98EPTB (192) 219 (58.6) 155 (41.4) 112 (58.3) 80 (41.7) 1.00 (0.70–1.42) 0.978






TB, tuberculosis; PTB, pulmonary tuberculosis; EPTB, extrapulmonary TB. Severe TB: DTB and TBM; Other TB: other than severe TB.0, wild-type genotype; 1, heterozygous genotype; 2, mutation genotype.An application of the Bonferroni correction for multiple testing (7 markers/tests) reset the significance threshold from 0.05 to 0.0125. Significant differences of the test forhomogeneity were indicated by a P value <0.05.

a ORs and P-values were adjusted for gender and age in control group vs. PTB group, control group vs. EPTB group, control group vs. other TB and control group vs. severe TBgroup in a logistic regression model.

b P value of the test for homogeneity between stratum-related ORs for genotypes of SNPs.

Table 4Distribution of IL-4 gene haplotypes in control vs. EPTB and control vs. severe TB in the Han Chinese pediatric population.

Gene (SNP) haplotypes Characteristics Control group (n = 374) TB group (n = 346) OR (95% CI)a P valuea

IL4 (rs2243268 + rs2243274)CA EPTB 561 (75.0) 316 (45.7) 1.52 (1.11–2.07) 0.008

severe 561 (75.0) 216 (31.2) 1.61 (1.14–2.26) 0.006

AG EPTB 183 (24.5) 68 (9.8) 0.66 (0.48–0.90) 0.008severe 183 (24.5) 53 (7.7) 0.62 (0.44–0.87) 0.006

Significant differences were indicated by a P value <0.05.a Chi-square tests analyze differences of haplotype frequencies of SNPs between control and EPTB or between control and severeTB.


Vidyarani et al., 2006). However, associations between susceptibilityof pediatric TB and SNPs of IL-4 gene have not been detected in anypopulation of children. Although 90% of MTB-infected individuals

are able to resist an overt development of the disease, which ismanifested only in the form of a latent infection, children tend tobe relatively more susceptible, and young children are highly likely

Fig. 1. LD plot of 4 SNPs in the IL-4 gene in Chinese Han population. The LDsdisplayed were calculated with the use of the r2 statistic in 753 subjects from theChinese Han population. SNPs with MAF of 5% or more were analyzed, rs2243250,rs2243268, rs2243274 and rs2243282. Rs2243268 and rs2243274 are in close LD.


to develop active TB after infection (Perez-Velez and Marais, 2012).The mechanisms by which children become more susceptible todeveloping active PTB and EPTB after exposure are an active areaof research. Young age and HIV coinfection are thought to be themost important risk factors for severe TB (Cotton et al., 2008; Wallsand Shingadia, 2004). The risk of EPTB increases as the degree ofimmunosuppression increases (Golden and Vikram, 2005). Thedata collected here are the first report of any association betweenpolymorphisms in any gene with predominant resistance to EPTBand severe TB in children, as opposed to PTB and other forms ofTB. Adult patients with sputum smears positive for PTB are

Fig. 2. Ex vivo whole-blood cytokine response and genotypes of IL-4 SNP rs2243268. WhCells were diluted 1:5 by RPMI 1640 and stimulated with 7.5 � 103/lL inactivated H37production by Milliplex assay. A: the level of IL-10 in supernatant of cells stumiated by Hvalues are noted by P value <0.05 and were calculated using the unpaired t-tests.

reported to express more IL-4 in the venous blood than EPTBpatients (He et al., 2010). However, the mechanisms by which IL-4 affects TB type and severity are not clear. One alternative expla-nation for the present data is that rs2243268 and rs2243274 areassociated with the expression level or the functions in IL-4 thatinfluence differentiation and cytokine (such as IL-10) secretion ofTh2 cells. The amount of cytokines produced by Th2 cells changescan cause cytokine secretion by other immune cells to change.

Because rs2243268 and rs2243274 are in complete LD (Fig. 1),we sequenced one of them (rs2243268) for exploring their mech-anism exerted on immune response. After stimulation with inacti-vated H37Rv, samples from children with the AA + AC rs2243268genotypes had significantly less IL-10 production and more IL-17A production than those with the CC genotype. These resultssuggest that children with mutated alleles of rs2243268 haveweaker anti-inflammatory response from Th2 cells and more pow-erful response from Th17 cells. In immune responses for MTB, Th2cells can inhibit functions of Th1 cells and macrophages by produc-ing cytokines (such as IL-4 and IL-10) (Hussain et al., 2002;Wangoo et al., 2001). In addition, the differentiation of Th17 canbe also inhibited by Th2 cytokines (Stockinger and Veldhoen,2007). As a result, weaker anti-inflammatory function of Th2 cellsmay be associated with more powerful cellular immune response.Th17 cells modulate the immune response to MTB by producing IL-17A, which is mostly associated with the induction of proinflam-matory programs associated with chemokine secretion andneutrophil recruitment (Torrado and Cooper, 2010). However,additional analysis is needed to confirm these speculations.

Rs2243250 of the IL-4 gene was found to be associated withadult TB in Iranian and Indian individuals. In Iranians, C alleleand the T/C genotypes were found significantly more common inTB patients than in controls; a similar study in Indians found thehigher frequency of CT genotype in PTB suggesting an associationbetween IL-4 rs2243250 and susceptibility to TB (Amirzargaret al., 2006; Vidyarani et al., 2006).The associations of SNPs ofthe IL-10 gene and susceptibility to TB have also been studied inEgyptians and Indians. In Egyptians, a statistically significant dif-ference in the frequency of GG genotype of rs1800896 SNP of IL-10 gene was found between patients with PTB and EPTB; inIndians, rs3024498 SNP of IL-10 gene did not show any strong rel-evance to immunity to TB (Abhimanyu et al., 2011; Amirzargaret al., 2006; Mosaad et al., 2010). However, statistical differencesamong the allelic and genotypic distributions of rs2243250,rs2243282, and SNPs of IL-10 gene were not observed in controlvs. TB or TB subgroup in the present study. The difference observedbetween different studies may be due to genetic differences of thestudied populations and differences in the study design.

Although the present findings offer new ideas for the diagnosis,prevention, and treatment of pediatric TB in China, the present

ole blood was harvested from individuals with rs2243268 genotypes AA, AC or CC.Rv. Cells were stimulated for 20 hours and supernatants were assayed for cytokine37Rv. B: the level of IL-17A in supernatant of cells stumiated by H37Rv.Significant P


work still has limitations: (1) because the level of IL-4 was lowerthan minimum detectable concentration (4.5 pg/mL) we did notprecisely measure the level of this cytokine; (2) we will sequencers2243283 (the other tag SNP of IL-4 gene) in a larger sample inour further study and we will try to collect the larger samples toverify our results of cytokine assays in the future.

5. Conclusions

The major findings of this pediatric study of SNPs of IL-4 and IL-10 genes were as follows: (i) no significant differences in allelic orgenotypic distributions of SNPs of IL-4 and IL-10 genes wereobserved between the control and TB groups; (ii) rs2243268-Aand rs2243274-G of the IL-4 gene were associated with a reducedrisk of developing EPTB and severe TB; (iii) the haplotype compris-ing alleles of rs2243268-A and rs2243274-G was found to be aresistance factor for EPTB and severe TB; (iv) an ex vivo studysuggested that children with mutated alleles of rs2243268 haveweaker anti-inflammatory functions of Th2 cells and more power-ful responses from Th17 cells.

Acknowledgements

The inactivated MTB H37Rv was kindly provided by ProfessorKanglin Wan in National Institute for Communicable Disease Con-trol and Prevention, Chinese Center for Disease Control and Pre-vention. We thank the staff of pneumology department in BeijingChildren’s Hospital for their help in our study. This work was finan-cially supported by grants from the Beijing Natural Science Foun-dation (No. 7121007) and National Natural Science Foundation ofChina (No. 30901632), Beijing Post-doctoral Research Foundationand China Postdoctoral Science Foundation.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.meegid.2014.01.031.

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rs2243268 and rs2243274 of interleukin-4 (il-4) gene are associated with reduced risk for...

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