Association Of IL-4 And IFNγ Gene Polymorphisms With The Susceptibility Of Respiratory Syncytial Virus Bronchiolitis

Background and ObjectiveRespiratory syncytial virus RSV bronchiolitis is the most common viral respiratory disease in infants and children.It is seasonal,peaking in the cold months.The morbidity is about 70%in infant,1%-2%infants will develop to severe cases.The more severe case will lead to die.The mild infection will pass,but severe RSV bronchiolitis is often followed by postbronchiolitic wheezing,cough.Risk factors for severe RSV disease include premature birth,concurrent heart or lung disease.But some healthy infants also will develop to severe RSV infection.Unfortunately,no safe and effective RSV vaccine is available.More data shows the host immune response plays the important role in the pathogenesis.When the infant suffered RSV,a lot of Th1/Th2 cytokines will be secreted.These cytokines include IL-10,IL-4,and IL-5 et al.Cross-regulation occurs among these Th1/Th2 cytokines.The response deviated toward Th2 is associated with severe disease.But some data are conflicting.The pathogenesis of RSV bronchiolitis is Th1/Th2 imbalance.Cytokine level is related to the gene polymorphism.Hull found bronchiolitis risk is linked to polymorphisms in the wide-spectrum chemokine receptor CCR5 and theIL-8,IL-10 locus.To our knowledge,no article has improved the relationship between Th1/Th2 cytokines polymorphism and RSV bronchiolitis. Owe to the different race,gene distribution is not the same.The objective of this research is to explore the association of Th1/Th2(IFNγγ/IL-4) cytokines polymorphism and haplotype with the susceptibility of respiratory syncytial virus(RSV)bronchiolitis.Subjects and MethodsThis study included 168 hospitalized patients aged＜2 years who suffered RSV bronchiolitis(RSV group),215 hospitalized patients under 2 years who suffered pneumonia but not RSV infection(Non-RSV group)and 88 healthy children under 2 years(control group).Infants who suffered bronchopulmonary dysphasia,premature birth,congenital heart disease,and underlying defects in immune or be given immunosuppression or corticosteroid were excluded. The genome DNA was extract in all subjects.A TaqMan fluorescence polymerase chain reaction in the IL-4-589 and -33 loci were tested in all subjects.IFNγCA short tandem repeat in all enrolled children were defined by capillary electrophoresis.IL-4 and IFNγ,serum level were measured by ELISA for 50 children in RSV group and 50 infants in Non-RSV group.Respiratory score was chosen to evaluate illness severity of RSV infection.Haplotypes were inferred by using of computer program PHASE. Linkage disequilibrium(LD)was measured between IL-4 gene 2 loci by using LDA software.Results1.The level of IL-4(pg/ml)in RSV group(M=0.84,P5=0.12,P95=12.5)was significant higher than Non-RSV group(M=0.29,P5=0.10,P95=6.29),(z=2.465, p=0.014).The IL-4 concentration from the infants bearing -589TT genotype (M=0.87,P5=0.13,P95=6.94)was significant higher than the infant with -589CT genotype.(M=0.77,P5=0.11,P95=11.95),(z=2.357,p=0.018).The infants with -33TT genotype expressed similar IL -4 concentration as comparing with -33CT genotype.(-33TT;M=0.84,P5=0.14,P95=7.21);(-33CT;M=1.57,P5=0.08, P95=12.5),(z=0.088,p=0.93)。2.IFNγ's level(pg/ml)was as follow;RSV group(M=6.22,P5=0.66,P95=61.40); Non-RSV group(M=10.07,P5=1.46,P95=96.66),the difference was significant. (z=2.096,P=0.036).The infants whose IFNγ,CA repeat were12 times had high IFNγ,level than non-12 times.A significant difference was found(p＜0.05).3.The frequency of CC,CT,TT genotype at IL-4 gene -589 sits were 1.2%,21.4%,77.4%in RSV group,and 1.9%,33%,65.1%in Non-RSV group,in control group the frequency was 1.1%,36.4%,62.5%respectively.A significant difference was found between RSV group and Non-RSV group.(X²= 6.821,p=0.033);When RSV group was compared with control group,a significant difference can be found as well(X²=6.621,p=0.037).There was no significant difference between Non-RSV group and control group(X²=0.47,p=0.791).4.The T allele of-589 loci was found in a significantly higher frequency among RSV children(88.1%)than in the Non-RSV group(81.6%)and control group (80.7%).On the contrary,C allele in RSV infant(11.9%)was significantly lower than infant in the Non-RSV group(18.4%)(X²=6.012, p=0.014,OR-C=0.6,95%CI;0.398-0.905,OR-T=1.666,95%CI;1.105-2.511),and control group(19.3%)(X²=5.134,p=0.023).There was no significant difference between Non-RSV group and control group(X²=0.074,p=0.786).5.The frequency of CC,CT,TT genotype at IL-4 gene -33 sits were 1.2%,23.2%, 75.6%in RSV group,and2.3%,34%,63.7%in Non-RSV group,in control group the frequency was 2.3%,39.8%,58%respectively.A significant difference was found between RSV group and Non-RSV group(X²=6.313,p=0.043);There was significant difference between RSV group and control group.(X²= 8.495,p=0.014).The result was not statistically significant between Non-RSV group and control group(X²=0.929,p=0.629).6.Frequency distribution of IL-4 -33 allele in three group were C allele 12.8%, 19.3%,22.2%,T allele 87.2%,80.7%,77.8%respectively.There was significant difference in allele frequency distribution between RSV group and No-RSV group(X²=5.807,p=0.016,ORC=0.614,95%CI;0.411～0.915,ORT=1.63, 95%CI;1.093～2.431);The frequency did not differ significant between Non-RSV group and control group.(X²=0.634,p=0.426)。7.The frequency of(CA12)+/(CA12)+,(CA12)+/(CA12)-,(CA12)-/(CA12)- in RSV group were 8.93%,58.33%,32.74%respectively.The frequency of Non-RSV group were19.53%,52.09%,28.37%respectively.A significant difference was found in the frequency distribution between two groups(X²= 8.392,p=0.015).Frequency of IFNγCA repeat 12 times was 38.10%in the RSV group which was significantly less than in the Non- RSV group for 45.58%(X²= 4.331,p=0.037).8.The RSV infant bearing -589 TT genotype had high respiratory score than infant with -589 CT genotype(3.02±0.44;2.81±0.46,t=2.56,p=0.01).The respiratory score in -33TT genotype infant was Significant higher than -33CT genotype infant (3.02±0.44;2.79±0.48,t=2.835,p=0.005).The respiratory score was 2.91±0.46 in the patient who had 12 times CA repeat and 3.0±0.43 for the patient who had Non-12 times CA repeat.The difference was significant(t=2.005,p=0.047).9.There is tight linkage disequilibrium between 2 loci of IL-4(-33C/T,-589C/T) (D'=0.719±0.0523,r2=0.4035).After reconstruction of the 2 loci of IL-4 (-33C/T,-589C/T),the frequency of TT haplotype constituted by IL-4-33 locus and IL-4-589 locus had significant difference between RSV group(80.65%)and Non- RSV group(70.47%),(p=0.001,OR_TT=0.572,95%CI;0.407～0.805).Other haplotypes in two groups had no significant difference.The frequency of combined genotype IFNγ(CA12)^-/(CA12)^-/IL-4—589TT/—33TT in RSV group(26.79%)was significant higher than in Non-RSV group(17.21%),(p=0.023, OR=0.568,95%CI;0.347～0.929)).Conclusions1.The IL-4 serum level in RSV group was higher than Non-RSV group,while IFN-γlevel was less than Non-RSV group.These finding suggest Th2 type response wais dominant in RSV bronchilitis.IL-4-589 TT genotype was a high IL-4 expressing genotype.If the patient had 12 times IFNγCA repeat,the IFNγlevel would be higher than Non-12 times.2.A significant association was revealed between polymorphism of IL-4 gene -33,-589 locus and RSV bronchilitis.TT genotype and T allele maybe susceptible to RSV bronchilitis.3.IFNγCA short tandem repeat was associated with the susceptibility of RSV bronchiolitis.CA short tandem repeat of Non-12 times maybe the susceptible genotype for RSV bronchiolitis.4.The respiratory score of IL-4-33TT type and IL-4-589 type were higher than CT type of these two loci.It can be inferred the RSV infants who had TT genotype were more severe than CT genotype.The respiratory score was high in infant who had Non-12 times CA repeat,the severity of RSV would be severe as well.5.There was tight linkage disequilibrium between the 2 loci of IL-4 (-33C/T,-589C/T). TT haplotype maybe the susceptible genotype for RSV bronchiolitis,the combined genotype IFNγ(CA12)—/(CA12)-/IL-4—589TT/—33TT will be as well.