Methyl-cpg-binding Domain Protein 2 Silencing Inhibits Th17 Differentiation of CD4+T cells Induced by Ovalbumin.

Background: Little is known about MBD2's epigenetic regulation in the immune pathogenesis of CD4+T cell differentiation. Objective: This study attempted to explore the mechanism of methyl-cpg-binding domain protein 2 (MBD2) in CD4+T cell differentiation stimulated by environmental allergen ovalbumin (OVA). Methods: Mononuclear cells were separated from the spleen tissues of male C57BL/6 mice. The OVA interfered with the differentiation of splenic mononuclear cells and CD4+T cells. The CD4+T cells were obtained by magnetic beads and identified by CD4 labeled antibody. CD4+T cells were transfected with lentivirus to silence MBD2 gene. A methylation quantification kit was used to detect 5-mC levels. Results: The purity of CD4+T cells reached 95.99% after magnetic beads sorting. Treatment with 200 µg/mL OVA stimulated the CD4+T cells differentiation to Th17 cells and promoted the secretion of IL-17. After being induced, the Th17 cell ratio increased. 5-Aza inhibited the Th17 cell differentiation and the IL-17 level in a dose-dependent manner. Under the intervention of the Th17 induction and 5-Aza, MBD2 silencing inhibited the differentiation of Th17 cell, and decreased the IL-17 and 5-mC levels in the cell supernatants. MBD2 silencing reduced the scale of the Th17 cell and IL-17 levels in the OVA-treated CD4+T cells. Conclusion: MBD2 affected IL-17 and 5-mC levels by mediating the Th17 cell differentiation in splenic CD4+T cells that were interfered with 5-Aza. OVA induced Th17 differentiation and increased IL-17 levels, inhibited by MBD2 silencing.

C-EBPß mediates in cigarette/IL-17A-induced bronchial epithelial-mesenchymal transition in COPD mice.

BACKGROUND: Cigarettes smoking and IL-17A contribute to chronic obstructive pulmonary disease (COPD), and have synergistical effect on bronchial epithelial cell proliferation. CCAAT/enhancer-binding protein ß (C-EBPß) could be induced by IL-17A and is up-regulated in COPD. We explored the effect of cigarettes and IL-17 on bronchial epithelial-mesenchymal transition (EMT) in COPD mice and potential mechanism involved with C-EBPß in this study. METHODS: COPD model was established with mice by exposing to cigarettes. E-Cadherin, Vimentin, IL-17A and C-EBPß distributions were detected in lung tissues. Primary bronchial epithelial cells were separated from health mice and cocultured with cigarette smoke extract (CSE) or/and IL-17A. E-Cadherin, Vimentin and IL-17 receptor (IL-17R) expressions in vitro were assessed. When C-EBPß were silenced by siRNA in cells, E-Cadherin, Vimentin and C-EBPß expressions were detected. RESULTS: E-Cadherin distribution was less and Vimentin distribution was more in bronchus of COPD mice than controls. IL-17A and C-EBPß expressions were higher in lung tissues of COPD mice than controls. In vitro, C-EBPß protein expression was highest in CSE + IL-17A group, followed by CSE and IL-17A groups. E-cadherin expression in vitro was lowest and Vimentin expression was highest in CSE + IL-17A group, followed by CSE or IL-17A group. Those could be inhibited by C-EBPß silenced. CONCLUSIONS: C-EBPß mediates in cigarette/IL-17A-induced bronchial EMT in COPD mice. Our findings contribute to a better understanding on the progress from COPD to lung cancers, which will provide novel avenues in preventing tumorigenesis of airway in the context of cigarette smoking.

The roles of dipeptidyl peptidase-4 and its inhibitors in the regulation of airway epithelial-mesenchymal transition.

Objective: Dipeptidyl peptidase 4 (DPP4), also known as CD26, is a transmembrane glycoprotein with peptidase activity expressed on epithelial cells and some immune cells. It also occurs as a soluble form. Studies have revealed that the expression level of lymphocyte sCD26/sDPP4 was elevated in the asthmatic patients. Airway remodeling increases in asthma severity and these structural changes include, amongst others, the loss of epithelial integrity because of cell shedding, goblet cell hyperplasia, destruction of ciliated cells, and EMT. So we try to find whether sCD26/sDPP4 has a role in pathological/dysregulated transition from bronchial epithelial cells into fibroblasts cells in response to TGFß1 exposure in vitro. Therefore, our purpose in the present work was to identify the role of sCD26/sDPP4 in airway EMT regulation. Methods: The EMT cell model was established based on human 16HBE cells. The effects of sCD26/sDPP4 and its inhibitors on airway EMT and that of sCD26/sDPP4 on Th17/IL-17 and its role in airway EMT were investigated in vitro. Results: The mRNA and protein level of E-Cadherin decreased after the treatment of TGF-ß1 in 16HBE cells, while α-SMA was up-regulated. The level of E-Cadherin was significantly down-regulated after the sCD26/sDPP4 stimulation, and that of α-SMA was dramatically elevated. DPP4 inhibitors promoted the level of E-cadherin and inhibited that of α-SMA. Additionally, in the DPP4-treated IL-17 cells group, E-Cadherin was markedly down-regulated at the mRNA and protein level, while α-SMA was reversely up-regulated. Conclusion: The TGF-ß1-induced EMT of human bronchial epithelial cells could be promoted by sCD26/sDPP4. The suppression of EMT in human bronchial epithelial cells was achieved by DPP4 inhibitor, and the TGF-ß1-mediated EMT of human airway cells was promoted by the synergy of IL-17 and sCD26/sDPP4 in vitro.

Cigarette and IL-17A synergistically induce bronchial epithelial-mesenchymal transition via activating IL-17R/NF-κB signaling.

BACKGROUND: IL-17A directly induces epithelial-mesenchymal transition (EMT) in alveolar epithelial cells. It could coordinate with cigarette smoke extract (CSE) to promote proliferation of bronchial epithelial cells. In this study, we aim to explore the direct effect of IL-17A and CSE on EMT in bronchial epithelial cells. METHODS: Bronchial epithelial cells were isolated from C57BL/6 mice, and cocultured with CSE or/and IL-17A. E-cadherin and Vimentin expressions in cells were detected using immunofluorescence staining. IL-17R expression was detected using immunohistochemistry staining. NF-κB expression was assessed using western blotting. When NF-κB was inhibited by BAY 11-7821, expressions of NF-κB, E-cadherin and Vimentin were measured. RESULTS: The protein expression of E-cadherin in bronchial epithelial cells was lowest in CSE + IL-17A group, followed by CSE group. In contrast, the protein expression of Vimentin was highest in CSE + IL-17A group, followed by CSE group. Similarly, IL-17R and NF-κB expressions were highest in CSE + IL-17A group, followed by CSE group and IL-17A group. NF-κB inhibitor could inhibit the expressions of E-cadherin and Vimentin. CONCLUSIONS: Cigarette and IL-17A could synergistically induce EMT in bronchial epithelial cells through activating IL17R/NF-κB signaling. Our findings contribute to a better understanding in airway EMT and pathogenesis of respiratory diseases, which are involved IL-17A and cigarette smoking. Those will provide novel avenues in the immunotherapy of lung diseases.