Suppression Of Asthmatic Airway Inflammation By Der P2 Recombined BCG And The Possible Mechanisms

Asthma is a chronic disorder of the airways, and is characterized by reversible airflow obstruction, airway eosinophil inflammation, persistent airway hyperreactivity, and airway remodeling. The pathology in asthma occurs as a consequence of increased production of IL-4, IL-5, and IL-13 by allergen-specific CD4+ Th2 cells. In addition, the risk of developing asthma is directly related to the acquisition of immediate hypersensitivity to environmental allergens. Meanwhile, house dust mites are the most important sources of indoor allergens responsible for the development of asthma.A more effective and long-lasting therapeutic approach for asthma focuses on the development of allergen recombined vaccine strategies that alter the underlying immune response and convert detrimental allergic responses toward protective immune responses, thereby modifying the course of the disease. We previously have engineered recombined BCG that express Der p2 of house dust mites on the cell wall. Subsequently, we established that the Der p2 recombined BCG induced a shift from Th2 to Th1 response in naive mice. However, the regulatory role of the Der p2 recombined BCG in asthmatic animal model is still unknown.The bacillus Calmette-Guèrin (BCG) vaccine is the most widely used Th1-inducing vaccine. Several studies argued that BCG may be applied to allergy treatment through inducing an immune deviation from Th2 to Th1. However, recent several years some studies argued that mycobacterium vaccae can be used as adjuvant to induce Tregs and then suppress asthmatic airway inflammation, which is not related to immune deviation. So, the immune mechanisms may be responsible for the suppressive effect of BCG/rBCG on asthmatic airway inflammation remains to be further investigated.Notch signaling represents a highly conserved pathway regulating cell proliferation and differentiation. Interaction between Notch ligands and receptors triggers liberation of the Notch intracellular domain (NIC) into cytoplasm. NIC then translocates into nucleus, where it binds to and transcription factor RBP-J. RBP-J regulates the expression of multiple downstream genes including Hes family members. Hes proteins are suppressive bHLH molecules that repress many bHLH family transcription factors.Notch signaling regulates lineage commitment at various stages of T cell maturation. Several studies have implicated the participation of Notch signaling in Treg differentiation. But potential roles of Notch signaling in the development and functional maintenance of Tregs are still unclear. Foxp3 acts as a master molecule controlling the development and function of Tregs. Whether Notch signaling can directly regulate the expression of FOXP3 and the possible mechanisms are still unclear.The aim of this study is: 1) to investigate whether the Der p2 recombined BCG can regulate asthmatic eosinophil inflammation and explore the possible mechanisms; 2) to investigate whether Notch signaling can regulate the expression of Foxp3 and elucidate the regulatory mechanisms. Our main results are as follows:1. We demonstrated that Der p2 rBCG dramatically inhibited airway inflammation, lung tissue pathology, mucus production, and mast cell degranulation in asthmatic mice in OVA and Der p2 induced asthmatic animal model. Analysis of the expression of Th2 cytokines in BALF revealed that Der p2 rBCG reduced the production of IL-4, IL-5 and IL-13. Then, Der p2 rBCG can effectively down-regulate allergic Th2 response.2. Analysis of IFN-γ/IL-4 in BALF and IFN-γ/IL-4 mRNA in the lung tissue of asthmatic mice revealed that Der p2 rBCG vaccination can induce a shift from Th2 to Th1 response in asthmatic mice and altered the highly polarized type 2 cytokine environment both in local airway and in whole lung tissue.3. We found that Der p2 rBCG up-regulated the TGF-βlevel in BALF in asthmatic mice; The relative proportion and the absolute number of CD4+CD25+Foxp3+ T cells were all incremental after vaccination with rBCG; Der p2 rBCG induced a CD4+CD25+Foxp3+ T cell could suppress the proliferation of CD4+ effector cells in vitro in an antigen-specific way; CD4+CD25+Foxp3+ T cell induced by Der p2 rBCG, which were adoptively transferred into allergen-sensitized recipients, could migrate to sites of thoracic local draining LNs and suppress local airway inflammation. So, the suppression by Der p2 rBCG was also associated with immune suppress mediated by Der p2 rBCG induced Tregs.4. We identified RBP-J-binding site and Hes-binding sites in both mouse and human FOXP3 promoter through bioinformatic analysis. Indeed, using a luciferase reporter assay, we showed that overexpression of NIC resulted in activation of the FOXP3 promoter. DNA pull-down assays indicated that NIC-RBP-J complex can bind to the related site in FOXP3 promoter. Blockade of Notch signaling with GSI can down-regulate the expression of Foxp3. Thus, Notch signaling could directly regulate Foxp3 expression. However, the Notch might not be involved in the lineage determination during Treg development, because the binding of NIC-RBP-J with the FOXP3 promoter could also be detected in conventional CD4+CD25- T-cells.5. We constructed a series of reporter plasmids in which the RBP-J-binding site and the N-boxes in the FOXP3 promoter were disrupted by deletion and site-directed mutagenesis. The results confirmed that the NIC-RBP-J complex was a transactivator on one hand, but on the other hand, Hes1 appeared to be a repressor of the FOXP3 promoter. Consistently, in the transfection assay, at higher dose of NIC when the FOXP3 promoter was turned down, Hes1 expression was significantly induced. Therefore, Notch signaling might regulate the FOXP3 promoter in a bi-phasic manner: at low magnitude Notch signaling it activated the FOXP3 promoter through the NIC-RBP-J complex, but at high magnitude Notch signaling it repressed the FOXP3 promoter through Hes1. 6. Our ChIP assays results revealed that Hes1 bound to the N-box region of the FOXP3 promoter specifically in CD4+CD25- T-cells, but not in CD4+CD25+ Tregs. Meanwhile, reporter assays indicated that Hes1 can intensively repress FOXP3 promoter activity. Thus, Hes1 might be an important regulatory factor at the transcriptional level in the lineage determination of Tregs development. In summary, we demonstrated that Der p2 rBCG, which was constructed by ourselves, can effectively down-regulate allergic Th2 response. Immune deviation and immune suppression co-existed in the immunoregulation process by Der p2 rBCG. These results provided another feasible immunoregulatory treatment method for asthma and enriched the immunological pathogenesis of asthma. In addition, we found that Notch signaling might directly regulate the FOXP3 promoter activity in a bi-phasic manner through RBP-J- and Hes1- dependent mechanisms and Hes1 might be an important regulatory factor at the transcriptional level in the lineage determination of Tregs development. These results riched the basal theory of the regulation of lymphocytes'development by Notch signaling and would facilitate further studies in the developmental and differential mechanisms of nTregs.