The Impact Of Th17 And IL-17 On The Pathogenesis Of Childhood Asthma

PARTⅠPercentages and function status of circulating T help cell 17(Th17) in asthmatic childrenSectionⅠ. Evaluating the protocol of analysing human Th17 cell by Flow CytometryObjective: Many studies in animal model indicated the key role of T help cell(Th)-17 in the pathology of autoimmune diseases and allergic diseases. The study is to evaluate the protocol for analysing human Th17 cell by flow cytometry.Methods: The mononuclure cells were separated from peripheral blood of healthy donors and cultured with different concentration of PMA plus Ionomycin and Golgistop. Th17 was analyzed by flow cytometry at different time points. The expression of membrane CD69 and IFN-γproducing Th1 were detected as positive control for T lymphocyte activtion.Results: The condition for activation of Th17 and Th1 was not identical. Culturing peripheral blood mononuclure cells(PBMC) in the present of PMA with a concentration from 10-100μg/L plus Ionomycin and Golgistop, for 4-12 hours was the appropriate protocol for both Th17 and Th1 detection. Too much more stimulus or culturing time influenced the detection of Th17 rather than improved it.Conclusion: The protocol for analysing human Th17 by flow cytometry presented here can be referenced.SectionⅡ. Percentage and function status of circulating T help cell 17(Th17) in asthmatic childrenObjective: Th17 is a recently identified subset of Th cell characterized by production of interleukin (IL)-17, which mediate neutrophils accumulation. Neutrophils accumulation in the airway is a feature of asthmatic children.The study observing the percentage and function status of Th17 in peripheral blood of asthmatic children,was to explore the role of Th17 on the pathogenesis of childhood asthma .Methods: Thirty children with acute asthma exacerbation and twenty healthy individual as control were recruited. The percentages of Th17, Th1 and Th2 were deceted by flow cytometry; the levels of IL-17 and IFN-γin peripheral blood mononuclear cell (PBMC) cultureed supernatant and IL-17, IL-23, total IgE in serum were detected by enzyme linked immunosorbent assy (ELISA),mRNA expression was detected by real-time RT-PCR. Results: (1) In asthmatic and healthy children, there was no significantly difference of the percentages of CD4+IL-17+T [(1.25±0.66)% vs (1.27±0.66)% ,P>0.05] and CD4+IL-4+T [2.398±2.548 vs 2.517±1.257(P>0.05)] in CD4+T cell population;the percentages of CD4+IFN-γ+T in asthmatic children was significantly lower than that in control [15.79±7.479 vs 24.10±12.70(P<0.05)] (2) The levels of IL-17 in PBMC culture supernatant of asthmatic children was significantly lower than that in control [17.53(0-245.57)pg/mL vs 101.74(25.12-500.60)pg/mL , P<0.01); So was the IL-17 mRNA expression in PBMC(4.78(1.0-35.49 vs 8.26(4.40-49.43)P=0.05),and it was positively correlated with protein production(r=0.76 P=0.01). There was no significantly difference of IFN-γproduction in both group[3507±2788 pg/mL vs 3027±2737 pg/mL(P>0.05)]. IL-17 in serum was less than 2 pg/mL and the IL-23 was less than 15pg/mL in both groups. (3) Total serum IgE in asthmatic children was significantly higher than that in control [499.26(2.3-945.1) vs 54.57(1.7-318.26)IU/mL, P<0.001]; there was no significantly difference of IL-17 or IFN-γproduction in PBMC cultured supernatant between IgE positive(>150 IU/ml) and negative asthmatic children.Conclusion: The results demonstrated that the percentages of Th1 and the function of Th17 in asthmatic children were impaired in peripheral blood, while the mechanism remains to be further studied . The change of Th1 percentage and Th17 function may be associated with the pathogenesis of childhood asthma.PARTⅡThe polymorphisms of IL-17 G-152A and its association with pediatric asthma in Southwestern Han Chinese populationObjective: The interleukin(IL)-17 is linked to the pathology of asthma ; IL-17 gene located on chromosome 6p,the genomic region of which has been reported to be linked to asthma and asthma-related phenotypes. The study was to investigate the association between single nucleotide polymorphism (SNP) of IL-17 G-152A with childhood asthma in Southwestern Han Chinese population.Methods: Eighty-nine hospitalized children with bronchiolitis, 88 children with asthma and 184 healthy controls were recruited. Polymerase chain reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) was used to analyse the IL-17 G-152A polymorphisms (rs2275913).Results: (1)IL-17 G-152A polymorphisms were present in all groups and in Hardy-Weinberg equilibrium. The frequencies of allele was 41.6% (A) and 58.4% (G) in the healthy children, with a genotype AA frequency of 14.7%, AG frequency of 53.8%,and GG frequency of 31.5%.(2)The frequencies of allele A and genotype AA in asthma group(P=0.048, P=0.013, respectively) , particularly in allergic asthma(P=0.002, P=0.001, respectively) were significantly higher than those of that in control group. Carriers of the A allele had a 1.44-fold (95% confidence interval: 1.00-2.06) risk of developing asthma than those homozygous for the G allele. AA genotype had a 2.44-fold (95% confidence interval: 1.32-4.50) risk of developing asthma than other genotypes. The strength of associations was moderately higher by allergy comorbidity. (3)There was no significantly difference of average level of lung function,PC20 and serum total IgE among the different genotype groups,but the proportions of subjects with abnormal total serum IgE and FVC,F EV1 values were significantly higher in subjects with the genotype AA than in those with AG and GG genotypes. (4)IL-17 production in PHA-induced PBMC was not different among individuals with different genotypes .(5)When patients with bronchiolitis were typed for IL-17 G-152A polymorphism, no difference was observed compared with control. But a significantly higher Streptococcus pneumoniae detection rate was shown in subjects with homozygous A allele compared with subjects with AG and GG genotype (37.5% vs 4.5%; P<0.001). The length of hospital stay was significantly higher in subjects with homozygous A allele than in subjects with AG and GG genotype(8.2 vs 6.3 days; P=0.02). Course of wheezing in subjects with homozygous G allele was significantly shorter than in subjects with AA and AG genotype ( 9.9 vs 11.3 days; P=0.03).Conclusion: These preliminary results suggested that IL-17 is a candidate gene that confers the genetic susceptibility for childhood asthma in Southwestern Han Chinese population. The homozygous -152A allele of IL-17 was associated with abnormal lung function and serum total IgE in asthmatic patients and with the clinical features of bronchiolitis, even though it seemed to have little influence on IL-17 production in PHA-induced PBMC. It demands further investigations to understand the function and mechanism of IL-17 G-152A polymorphisms in regulating IL-17 expression,as well as increasing samples and association of other SNPs of IL-17 with asthma.