| Part One:The Expression of REG3A, Inflammatory Reaction and Destruction of the Lung with S. aureus Infection in Patients with AD-HIESObjective:This study aimed to clarify the expression of REG3A, inflammatory reaction and destruction of the lung with S.aureus infection in patients with AD-HIES, and to further explore the mechanism of the severe destruction of the lung. In addition, to study the effect of STAT3 gene mutation on the expression of IL-17% IL-22 and REG3A of the lung in patients with AD-HIES.Methods:The clinical data about pneumonia, pathogenic bacteria and complications of the lung of 10 patients with AD-HIES was statistically analyzed. The structural changes of the lung were detected by HE staining. The expression levels of Th17 cell associated cytokines in plasma of patients with AD-HIES were tested with Bio-Plex suspension system. The numbers of Th17 and IL-17+γδT cells in peripheral blood of patients with AD-HIES were detected by flow cytometry. The expression of p-STAT3, IL-17, IL-22 and REG3A in the lung of AD-HIES patients were observed by using immunohistochemistry.Results:All 10 patients had recurrent pneumonia,7 patients had typical S.aureus pneumonia,5 patients accompanied with pneumatoceles,2 patients received lung surgery. HE staining of lung sections showed that the structure was severely destroyed, less neutrophil and much eosinophil was observed in the lung. The concentration of IL-17 was decreased, and IL-6 was increased in the plasma of patients with AD-HIES compared to the healthy control children by using Bio-Plex suspension system. However, there was no significant difference of the concentration of IL-22 in the plasma between AD-HIES patients and healthy control children. The numbers of Th17 and IL-17+γδT cells were significantly decreased in peripheral blood of patients with AD-HIES. Immunohistochemistry showed that the expression levels of p-STAT3, IL-17, IL-22 and REG3A in the lung of AD-HIES patients were significantly decreased.Conclusions:Patients with AD-HIES were susceptible to suffer pneumonia from the young, especially S.aureus pneumonia. The structure of the lung was severely destroyed due to S. aureus infection, suggesting that the repair function of the lung in patients with AD-HIES was defective. The decreased numbers of Th17 and IL-17+γδT cells and low expression levels of p-STAT3, IL-17, IL-22 and REG3A in the lung maybe the critical reasons for recurrent S. aureus infection and defective repair function of the lung in patients with AD-HIES.Part Two:AD-HIES Linked STAT3 Gene Mutation Inhibit the Expression of REG3A in the Bronchial Epithelia 16HBE CellsObjective:This study aimed to investigate the effects of AD-HIES linked STAT3 gene mutation (R382W, V637M and T620S) on the expression of REG3A in bronchial epithelia cells. In addition, to further explore the factors and regulatory mechanism of promoting REG3A expression.Methods:16HBE cells were transfected with one of the variants of STAT3-R382W, STAT3-V637M, STAT3-T620S and STAT3-Wt, then the expression of STAT3 was confirmed using Western blot. We observed the changes of REG3A, IL-6, IL-17and IL-22 expression levels in the 16HBE cells with S.aureus infection using Real-time PCR. The change of REG3A expression level in 16HBE cells was detected with Real-time PCR and Western blot following treatment of different concentrations of IL-6, IL-17 or IL-22. In the last, to compare the effects of various STAT3 mutants on the expression levels of REG3A in 16HBE cells treated with S.aureus, IL-17 or IL-22, the change of REG3A protein expression was detected by Western blot.Results:S.aureus infection significantly increased the levels of REG3A and IL-6 mRNA expression. However, there was no expression of IL-17 and IL-22 in 16HBE cells. Meanwhile, the levels of REG3A, STAT3 and p-STAT3 protein expression were significantly increased in 16HBE cells with the challenge of S.aureus. However, STAT3 mutants including R382W, V637M and T620S, obviously inhibit the expression of REG3A in 16HBE cells with the challenge of S.aureus. To further study, we found that STAT3 mutants also inhibit the phosphorylation of STAT3, then lead to low expression of REG3A. The results of Real-time PCR showed that 250 ng/ml IL-6,50 ng/ml IL-17 or 400 ng/ml IL-22 could obviously up-regulate the expression of REG3 A. However, STAT3 mutants inhibit the expression of p-STAT3 in 16HBE cells with the treatment of IL-17 and IL-22.Conclusions:S.aureus and certain concentration of IL-6, IL-17 and IL-22 can up-regulate the expression of REG3A through inducing STAT3 phosphorylation. However, AD-HIES linked STAT3 gene mutations inhibit STAT3 phosphorylation due to its dominant negative function, and further inhibit REG3A expression.Part Three:REG3A Improves the Proliferation of 16HBE Cells with AD-HIES Linked STAT3 Gene Mutation through PI3K/AKT Signaling PathwayObjective:This study aimed to clarify the role of REG3A on improving proliferation of 16HBE cells, and to explore the changes of critical proteins associated with PI3K/AKT signaling pathway in improving proliferation of 16HBE cells.Methods:16HBE cells were transfected with one of the variants of STAT3-R382W, STAT3-V637M, STAT3-T620S and STAT3-Wt, then treated with recombinant REG3A, IL-17 or IL-22, respectively. The proliferation of 16HBE cells with STAT3 gene mutation was detected by Cell Counting Kit-8 experimental method. In addition, after treatment with PI3K inhibitor (Wortmannin) and recombinant REG3A, by combine or separately, the proliferation of 16HBE cells was also detected by Cell Counting Kit-8. EXTL3 which is REG3A receptor, and critical proteins of PI3K/AKT signaling pathway were detected in 16HBE cells with STAT3 gene mutation following treatment with recombinant REG3A, using Western blot.Results:Recombinant REG3A significantly improved the proliferation of 16HBE cells with STAT3 gene mutation, suggesting that REG3A improves the proliferation of 16HBE cells independent of STAT3 signaling pathway. However, there was no significant effect of IL-17 and IL-22 on the proliferation of 16HBE cells with STAT3 gene mutation. In addition, the proliferation of 16HBE cells was significantly decreased after treatment with PI3K inhibitor (Wortmannin) combined with recombinant REG3A compared to the group with recombinant REG3A treatment alone, suggesting that REG3A improves proliferation of 16HBE cells through PI3K pathway. Meanwhile, recombinant REG3A significantly increased the expression of EXTL3. The level of p-AKT protein expression was significantly increased in 16HBE cells with STAT3 gene mutation following treatment with recombinant REG3A, as shown by Western blot. However, there was no significant effect of IL-17 and IL-22 on the expression of p-AKT in 16HBE cells with STAT3 gene mutation.Conclusions:REG3A improves the proliferation of 16HBE cells with AD-HIES linked STAT3 gene mutation (R382W, V637M and T620S) through acting on EXTL3 receptor and PI3K/AKT signaling pathway; IL-17 and IL-22 have no significant influence on the proliferation of 16HBE cells with STAT3 gene mutation, suggesting that REG3A may have some therapeutic effects on improving lung tissue repair of patients with AD-HIES through promoting proliferation of respiratory epithelial cells, which is independent of STAT3 pathway. |
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