The Role Of PTEN Gene And Th1/Th2Cytokines In Children Asthma

Background:Bronchial asthma (also called asthma) is a common disease in the world and the incidence is gradually rising. It is the most common chronic lung disease which is a threat to the public health. The incidence of asthma is the highest among the child bronchial illnesses in the developed countries. There are about300million patients with asthma around the whole world, according to the report from WHO. The incidence and mortality of asthma is increased year by year. There are more than30million patients in China and the mortality of asthma is the highest in the world. Bronchial asthma is now a burning question on the public health.Asthma is influenced by immune, genetic and environmental factors and the mechanisms of asthma have not been comprehensively reviewed till now. The researches on asthma before were mainly about allergic inflammation induced by Th2cytokines. More attention is focused on the genetic etiology of asthma recently. Asthma has been shown to represent a multiple genetic illness. There are more than100genes reported associated with asthma, for instance,5q31、6p21、7q、11q12、11q13、12q13-24、13q14、20p13.PTEN gene (Phosphatase and tensin homolog deleted on chromosome10) was discovered in1997as the first dual-specificity phosphates activity of tumor suppressor gene. PTEN gene located at chromosome10q23.3,200kb long with9exons and8introns, and encoded a403-AA peptide. PTEN gene functions primarily as a lipid phosphatase and regulates cell growth, apoptosis, migration through the phosphatidylinositol-3-kinase (PI3K/Akt) signaling pathway. Genetic mutations have found on exons of PTEN gene except exon9. Exons5and8showed a higher mutation frequency than others. PTEN gene is one of the most frequently mutated tumor suppressors in human cancer. Thereby, study on PTEN gene and its signal transduction would be useful to treat the related diseases.The role of PTEN gene in allergic airway inflammation is paid more and more attention recently. Young et al. firstly described the involvement of PTEN in airway hyperresponsiveness and inflammation in a murine model of asthma. The findings have indicated that PTEN might play a pivotal role in the pathogenesis of asthma by decreasing IL-4, IL-5and eosinophil cationic protein levels. Another study has revealed that PTEN reduced VEGF (vascular endothelial growth factor) expression in allergen-induced airway inflammation. Adachi et al. have suggested that PTEN negatively regulates eosinophils (EOS) survival, chemotaxis and allergic inflammation. PTEN has a key role in lung stem cell homeostasis, cell differentiation, and consequently resistance to lung injury. A recent study has found that PTEN decreases the expression of Th2cytokine and has correlation with the balance of Thl/Th2. Dexamethasone (DXM) increases the expression of PTEN protein and inhibits the airway inflammation. A research in vitro has resulted in PTEN overexpression and inhibiting the proliferation of HASMCS (human airway smooth muscle cells) possibly through the PI3K/Akt pathways. At present, the investigations concerned the correlation of mutations of PTEN gene and bronchial asthma are rare either in domestic and foreign.Eosinophil response appears to be a critical feature in asthma. Eosinophil is the most reactive cell in allergic illnesses. Eosinophil accumulation and subsequent activation in bronchial tissues play vital roles in the pathophysiology of bronchial asthma. The role of eosinophils in allergic airway disease as being fundamentally active has been approved by animal models of genetic eosinophil depletion, human studies of eosinophil depletion with monoclonal antibody against IL-5. A series of investigations establish eosinophils as immunoregulator cells with key roles in Th2airway inflammation. Detection of level of EOS has an important clinical significance in asthma diagnosis, treatment and prognosis. The targeting of EOS may constitute a new strategy for the treatment of asthma.Asthma is characterized by chronic airway inflammation mainly of eosinophil, mast cell and T-helper cell (Th cell). CD4+T cell (also called Th cell), a subpopulation of T cell, a pivotal regulator in the pathophysiology of asthma. Antigen-activated CD4+T cells differentiate into Th1cytokines and Th2cytokines. They keep balancing in normal conditions. Recent researches have indicated that the role of Th1/Th2cytokine immune imbalance:Th1/Th2cytokine immune imbalance, causing injury of human immune function, is one of the critical mechanisms in asthma. The pathologic role of Th2cells is mediated through the release of Th2cytokines that are essential for immunoglobulin E synthesis, chemokine production, airway eosinophilia, smooth muscle hyperplasia, mucus production. Th1cells secrete interferon (IFN-γ) display a protective function in allergic asthma. IL-4is mainly secreted by Th2cells, involved in multiple inflammatory reactions, and plays an important role in an inflammatory immunoreaction. IL-5is a major regulator of eosinophil and has a direct relationship with the peripheral EOS count in asthma. IL-5, which is critical to recruitment, activation, chemotaxis and proliferation of eosinophil, causes allergic inflammation. IFN-γ, a typical Th1type cytokine, modulates the activation, differentiation and recruitment of EOS. IFN-γ induces the dominate differentiation of Thl cells, inhibits that of Th2cells and regulates Thl/Th2balance. This can reduce the incidence of asthma.Objectives:To explore the correlation between Phosphase and tensin homolog deleted on chromosome10(PTEN) gene and children bronchial asthma, to investigate the relationship of PTEN gene with the number of eosinophils (EOS), to study the role of Thl/Th2cytokines in asthma, which may provide some theoretical basis for mechanisms and management of asthma.Methods:1. Thirty children with asthma (clinical paracmasis), outpatients of our hospital from April1st,2010to August31st,2011, were enrolled as asthmatic group. Meanwhile, thirty age and sex matched healthy children were selected as control group. The diagnostic standard accords with bronchial asthma formulated by Respiratory group, Academy of Pediatrics, Chinese Medical Association (2008). All children without use of hormone and acute inflammation in one week are enrolled. Informed consent forms were signed by guardians.2.3ml peripheral vein blood was drawn from each child with empty stomach in the early morning,1ml of each blood sample was extracted genomic DNA. Other2ml of each one was prepared serum after preparation of blood smear.3. The mutations of exon5and8of PTEN gene were detected by polymerase chain reaction (PCR), gene clone and DNA sequencing and analysis.4. The expression content of IL-4, IL-5and IFN-y in serum was detected by enzyme-labeled immunosorbent assay (ELISA).5. The count of peripheral blood EOS was determined by high magnification. 6. Data are expressed as mean±SEM. Statistical comparisons were performed using t test and Wilcoxon rank sum test. Statistical significance was set at P<0.05. All statistical analysis was operated with SPSS13.0software.Results:1. No mutation was found at exon5and8of PTEN gene either in asthmatic group or in control group.2. The replacements of PTEN eighth intron (+32) T to G were detected in asthmatic group, and the mutation rate was86.7%(26/30).3. No mutation at intron8of PTEN was found in control group.4. The count of peripheral blood EOS in the asthmatic group420±126(X106/L) was higher obviously than that in the control group165±74(X106/L)(t=9.538, P<0.001)5. The count of peripheral blood EOS of asthmatic children with mutations was higher than that of asthmatic children without mutations (W=25.500, P=0.026)6. The level of IL-4of serum in asthmatic children158.23±28.76(ng/L) is higher remarkably than those of control group70.16±15.35(ng/L)(t=14.794, P<0.001)7. The level of IL-5of serum in asthmatic children45.32±12.15(ng/L) is higher remarkably than those of control group27.58±7.34(ng/L)(t=6.842, P<0.001)8. The level of IFN-y of serum in asthmatic children32.21±6.25(ng/L) is lower remarkably than that of control group53.65±13.19(ng/L)(t=8.047, P<0.001)Conclusions:1. No mutation was found at exon8of PTEN gene. The replacements of PTEN gene eighth intron (+32) T to G were detected.2. The count of peripheral blood EOS in the asthmatic group was higher obviously.3. The count of peripheral blood EOS of asthmatic children with mutations was markedly higher than that of asthmatic children without mutations.4. No mutation at exon5and intron5of PTEN gene was found.5. Asthma children have an imbalance on Thl/Th2cell immunity, that is hyperfunction of Th2cell immunity and hypofunction of Thl cell immunity.