ABSTRACT
BACKGROUND: Airway remodeling is one of the reasons for severe steroidresistant asthma related to HMGB1/RAGE signaling or Th17 immunity. OBJECTIVE: Our study aims to investigate the relationship between the HMGB1/RAGE signaling and the Th17/IL-17 signaling in epithelial-mesenchymal transformation (EMT) of airway remodeling. METHODS: CD4+ T lymphocytes were collected from C57 mice. CD4+ T cell and Th17 cell ratio was analyzed by flow cytometry. IL-17 level was detected by ELISA. The Ecadherin and α-SMA were analyzed by RT-qPCR and immunohistochemistry. The Ecadherin, α-SMA, and p-Smad3 expression were analyzed by western blot. RESULTS: The HMGB1/RAGE signaling promoted the differentiation and maturation of Th17 cells in a dose-dependent manner in vitro. The HMGB1/RAGE signaling also promoted the occurrence of bronchial EMT. The EMT of bronchial epithelial cells was promoted by Th17/IL-17 and the HMGB1 treatment in a synergic manner. Silencing of RAGE reduced the signaling transduction of HMGB1 and progression of bronchial EMT. CONCLUSION: HMGB1/RAGE signaling synergistically enhanced TGF-ß1-induced bronchial EMT by promoting the differentiation of Th17 cells and the secretion of IL-17.
ABSTRACT
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.
Subject(s)
DNA-Binding Proteins , Interleukin-17 , Th17 Cells , Animals , Male , Mice , Cell Differentiation , Epigenesis, Genetic , Interleukin-17/metabolism , Mice, Inbred C57BL , Ovalbumin/metabolism , DNA-Binding Proteins/geneticsABSTRACT
BACKGROUND: Dipeptidyl peptidase-4 (DPP4) is a transmembrane glycoprotein, prevalent across a variety of tissues and cells and can be foundin a solubilised in peripheral blood. This paper aims at determining the role of sCD26/sDPP4 in Th17 cell polarization and airway epithelial cell to epithelial mesenchymal transition (EMT) in asthma. METHODS: Female C57BL/6J mice were treated with ovalbumin to constructed asthma mice. The CD4+ T cell, and bronchial epithelial cells (BECs) were purified from the spleens and bronchus of mice. The KRT8 expression in BECs were identified by immunofluorescence (IF). Th17 cells were differentiated from a CD4+ T cell. Flow cytometry was usewd to identify and calculate the Th17 and Treg cells. Mice woth asthma were treated by DPP4 overexpressing lentivirus or DPP4 inhibitor. Histopathological modifications were assessed by hematoxylin-eosin (HE), periodic acid Schiff (PAS), and Masson staining. The total number of leucocytes was detected using a hemocytometer. For detection, quantitative Real-time PCR (qRT-PCR), western blotting (WB), and IF were used to evaluate the expression of E-cadherin and alpha-smooth muscle actin (α-SMA). Enzyme-linked immunosorbent assay (ELISA) was performed to analyze the DPP4, IL-4, IL-5, IL-13 and IL-17 levels. RESULTS: The findings suggest that sCD26/sDPP4 promote CD4+ T cells differentiation into Th17 cells in a depending on the applied dose. sCD26/sDPP4 up-regulated the expression of α-SMA and down-regulated the expression of E-cadherin in TGF-ß1-induced mouse BECs, which was reversed by DPP4 inhibitor. Co-culture induced a synergic effect between Th17 cells and sCD26/sDPP4 on the formation of airway EMT in BECs. Furthermore, DPP4 inhibitor prevented lung-bronchial inflammatory infiltration, mucus secretion, goblet cell hyperplasia and collagen deposition in asthma mice. Meanwhile, DPP4 inhibitor decreased the levels of DPP4, IL-4, IL-5, IL-13, IL-17 and increased the total number of leukocytes in bronchoalveolar lavage fluid of asthma mice. In addition, DPP4 inhibitor also inhibited airway EMT and Th17 cell polarization in asthma mice. CONCLUSIONS: The results in this paper show that up-regulation of DPP4 enabled airway inflammation and airway remodeling in asthmatic mice by modulating the Th17/IL-17 axis and accelerating the airway EMT, which isa therapeutic target in asthma.
Subject(s)
Asthma , Dipeptidyl Peptidase 4 , Epithelial-Mesenchymal Transition , Th17 Cells , Animals , Female , Mice , Asthma/chemically induced , Asthma/drug therapy , Asthma/metabolism , Cadherins , Dipeptidyl Peptidase 4/metabolism , Dipeptidyl-Peptidase IV Inhibitors/pharmacology , Disease Models, Animal , Interleukin-13/metabolism , Interleukin-17/metabolism , Interleukin-4/metabolism , Interleukin-5/metabolism , Lung/metabolism , Mice, Inbred BALB C , Mice, Inbred C57BL , Ovalbumin , Th17 Cells/metabolism , Th17 Cells/pathologyABSTRACT
Impressive achievements in clinical trials to treat hemophilia establish a milestone in the development of gene therapy. It highlights the significance of AAV-mediated gene delivery to liver. AAV5 is a unique serotype featured by low neutralizing antibody prevalence. Nevertheless, its liver infectivity is relatively weak. Consequently, it is vital to exploit novel AAV5 capsid mutants with robust liver tropism. To this aim, we performed AAV5-NNK library and barcode screening in mice, from which we identified one capsid variant, called AAVzk2. AAVzk2 displayed a similar yield but divergent post-translational modification sites compared with wild-type serotypes. Mice intravenously injected with AAVzk2 demonstrated a stronger liver transduction than AAV5, roughly comparable with AAV8 and AAV9, with undetectable transduction of other tissues or organs such as heart, lung, spleen, kidney, brain, and skeletal muscle, indicating a liver-specific tropism. Further studies showed a superior human hepatocellular transduction of AAVzk2 to AAV5, AAV8 and AAV9, whereas the seroreactivity of AAVzk2 was as low as AAV5. Overall, we provide a novel AAV serotype that facilitates a robust and specific liver gene delivery to a large population, especially those unable to be treated by AAV8 and AAV9.
