Association Study Of Transforming Growth Factor β1 With Asthma And Asthma-Related Phenotypes

Asthma is a complex disorder caused by interactions between multiple genes and has an important genetic component but no clear pattern of inheritance.It is most likely that any particular susceptibility variant has a relatively minor effect on the asthma phenotype and that the magnitude of its effect will be influenced by genes at other loci(gene-gene interactions) and by the environmental factors(gene-environment interactions). Descriptions of the disorder focus on characteristics of high serum IgE, airway eosinophils infiltration,mucosa hypersecreation and airway hyperresponsiveness in the presence of underlying chronic airway inflammation and airway remodeling.With partial apprehension,the concrete pathogenesis of asthma is unclear.Transforming growth factor-β1(TGF-β1)is a pleiotropic cytokine involved in asthmatic airway inflammation and immune response with its anti-and pro-inflammatory function.It is also believed to play an essential role in airway remodeling that occurs in asthmatic patients. Because of discrepant results derived from different studies,further investigation shoud be put forward to clarify the potential role of TGF-β1 in the pathogenesy of asthma.CD4⁺ CD25⁺ regulatory T cell exerts a vital role in maintaining immune homeostasis,via its cell-cell contact suppression involving in surface-bound TGF-β1.Up to now,rare data can be attained with respect to the relationship between surface-bound TGF-β1,expressed on CD4⁺ CD25⁺ regulatory T cell,and asthma or asthma-related phenotypes. TGF-β1,as an asthma candidate gene,has been extensively studied on the single nucleotide polymorphisms(SNPs)asociated with asthma and asthma-related phenotypes,however,with contradictory consequence in diverse populations.Currently,there is no report about the association of TGF-β1 SNP/haplotype with asthma or asthma-related phenotypes in a southeastern Chinese Han population,as well as the interactions between TGF-β1 and other genes.In view of these issues,we try to reveal the association of TGF-β1 between asthma and asthma-related phenotypes in a southeastern Chiese Han population and to illuminate the potential role of TGF-β1 in the pathogenesis of asthma.Part 1 Expression of membrane-bound TGF-β1 and other surface markers on peripheral CD4⁺CD25^highT cells in patients with atopic asthma:role of inhaled glucocorticoidsBackground:CD4⁺CD25⁺ regulatory T cells(Tregs)mediate immune suppression through cell-cell contact with surface molecules,particularly transforming growth factorβ1(TGF-β1),cytotoxic T lymphocyte-associated antigen 4(CTLA-4)and glucocorticoid-induced tumor necrosis factor receptor family-related protein(GITR),but little is known about the exact role of Tregs in the pathogenesis of asthma.Objective:To characterize the expression of surface markers on peripheral blood mononuclear cells(PBMC)-derived Tregs in patients with atopic asthma and healthy subjects,and to investigate the role of inhaled glucocorticoids on them.Methods:Expression of surface molecules on CD4⁺CD25^highTregs was detected by flow cytometry.The effect of inhaled glucocorticoids on expression of the surface molecules in Tregs was determined in vivo and in vitro.Total serum IgE and high-sensitivity C-reactive protein were measured by ELISA and latex enhanced immunoturbidimetric assay, respectively.Results:Equivalent numbers of peripheral Tregs were found in patients with atopic asthma(stable and acute)and healthy subjects.Tregs preferentially expressed CTLA-4,GITR,toll-like receptor 4(TLR4), latency-associated peptide(TGF-β1/LAP),and forkhead box P3(FOXP3). Patients with acute asthma had decreased numbers of CD4⁺CD25^highLAP⁺ T cells relative to healthy subjects and stable asthmatics.Inhaled glucocorticoids enhanced the percentage of Tregs expressing LAP in vivo and in vitro dose-dependently.Furthermore,the percentages of Tregs expressing LAP negatively correlated with total serum immunoglobulin E levels and severity of asthma,while positively correlated with forced expiratory volume in one second percentage of the predicted value (FEV₁%)in patients with asthma.Conclusion:Our data suggest that membrane-bound TGF-β1 is a potential candidate for predicting the severity of asthma,and may contribute to the sustained remission of asthma.Strategies targeting Treg cells on their surface markers,especially TGF-β1,are promising for future therapy of asthma.Part 2 Association of TGF-β1 polymorphisms with asthma risk and gene-gene interactions with CD14 Background:Asthma is a complex disorder caused by interactions between multiple genes of small to modest effect and has an important genetic component but no clear pattern of inheritance.As one of the potential candidate genes,transforming growth factorβ1(TGF-β1)was extensively studied on the association with asthma susceptibility. However,inconsistent results were obtained from studies in diverse populations.Objective:This study aimed to evaluate the effects of polymorphisms in TGF-β1 on asthma risk and gene-gene interactions with CD14 in a southeastern Chiese Han population.Methods:We consecutively recruited 318 unrelated adult asthmatic patients and 352 healthy volunteers from the same area of southeast China.Genotyping of each selected single nucleotide polymorphism (SNP)in TGF-β1 and CD14 was performed using SNPstream^(?)and TaqMan^(?)SNP genotyping technology.We conducted case-control association studies between the selected SNPs and asthma.Results:The results showed that alleles and genotypes of neither the 3 TGF-β1 SNPs(i.e.rs1800469,rs1982073 and rs12983047)nor CD14 SNP rs2563298 were separately found to be associated with asthma. TGF-β1 haplotype analysis confirmed these results.The carriers of TGF-β1 rs1800469 TT and CD14 rs2563298 CA/AA genotypes had a significantly decreased risk for asthma[adjusted odds ratio(OR)=0.19, 95%confidence interval(CI)=0.07-0.55],which showed blazing interactions between these two SNPs(adjusted OR=8.23,95%CI= 2.68-25.30).Likewise,such interactions were also found between rs1982073 and rs2563298.Conclusion:In our southeastern Chinese Han population,the TGF-β1 and CD14 genes show an epistatic effect on the risk of asthma.It could be clinically useful both for identifying patients at risk of asthma and for preventing its occurrence.Part 3 Association of TGF-β1 polymorphisms with astnma-related phenotypesBackground:Although our study suggests that polymorphisms in TGF-β1 are not associated with athma risk in a southeastern Chinese Han population,some investigations demonstrate genetic variants of TGF-β1 are related to asthma-related phenotypes.Objective:This study aimed to evaluate the effects of polymorphisms in TGF-β1 on asthma-related phenotypes,namely,proportion of peripheral CD4⁺CD25^highLAP⁺ T cells,total plasma IgE level,plasma TGF-β1 concentration,eosinophil counts,pulmonary function,SPT and asthma severity degrees.Methods:We consecutively recruited 318 unrelated adult asthmatic patients from the same area of southeast China.Genotyping of each selected single nucleotide polymorphism(SNP)in TGF-β1 was performed using SNPstream^(?)and TaqMan^(?)SNP genotyping technology. Peripheral CD4⁺CD25^highLAP⁺T cells were detected by flow cytometry. Total plasma IgE and plasma TGF-β1 concentration were measured using ELISA.We conducted case only association studies between the selected SNPs and asthma-related phenotypes.Results:The carriers of TGF-β1 rs1800469 CC genotype had a significantly increased proportion of peripheral CD4⁺CD25^highLAP⁺ T cells,as compared with those of TT or TC(P＜0.001).And this trend was also found in the TGF-β1 rs1982073 TT carriers.FEV₁/FVC was marginly higer in the carriers of TGF-β1 rs12983047 AG/GG than in AA (P=0.034).No association was found between the three SNPs and other asthma-related phenotypes.Conclusion:Our results reveal that polymorphisms in TGF-β1 are associated with the proportion of peripheral CD4⁺CD25^highLAP⁺ T cells in asthmatics but not other asthma-related phenotypes.Polymorphisms in TGF-β1 have a critical role in the pathogenesis of asthma,presumably through affecting the expression of membrane-bound TGF-β1/LAP on CD4⁺CD25^highregulatory T cells.