EPO modified MSCs can inhibit asthmatic airway remodeling in an animal model.

There was no effective measures can be obtained at present to reverse or prevent airway remodeling. We investigated the therapeutic effect of Erythropoietin (EPO) gene modified mesenchymal stem cells (MSCs) on asthmatic airway remodeling and the possible underlied molecular mechanisms. EPO gene was transfected into MSCs via lentivirus vector. The transfected cells (EPO-MSCs) were identified by flow cytometry and the EPO secreting function was detected by PCR and Western blot. MSCs or EPO-MSCs were administrated to albumin (OVA)-induced chronic asthmatic mouse model via tail veins. The asthmatic phenotype was analyzed. Number of cells in bronchoalveolar lavage fluid (BALF) was counted using a hemocytometer. Histological findings of airways were evaluated by microscopic examination. The concentrations of interleukin 4(IL-4), interleukin 5(IL-5), and interleukin 13(IL-13) in lung homogenate were determined by ELISA. The activation state of transforming growth factor-ß 1 (TGF-ß1), Transforming growth factor beta-activated kinase 1 (TAK1), and p38 Mitogen Activated Protein Kinase (p38MAPK) signaling was detected by Real-Time PCR and Western blotting. EPO-MSCs were successfully constructed. EPO-MSCs showed a more potently suppressive effect on local asthmatic airway inflammation and the level of IL-4, IL-5, and IL-13 in lung tissue than MSCs. Moreover, the numbers of goblet cells, the thicknesses of smooth muscle layer, collagen density, percentage of proliferating cell nuclear antigen positive (PCNA+ ) mesenchymal cells, and von Willebrand factor positive(vWF+ ) vessels were also significantly inhibited by EPO-MSCs. Furthermore, EPO-MSCs could downregulate the expression of TGF-ß1, TAK1, and p38MAPK in lung tissue both in mRNA level and in protein level. EPO gene modified MSCs may more efficiently attenuate asthmatic airway remodeling, which maybe related with the downregulation of TGF-ß1-TAK1-p38MAPK pathway activity.

Der p2 recombinant bacille Calmette-Guerin priming of bone marrow-derived dendritic cells suppresses Der p2-induced T helper17 function in a mouse model of asthma.

BACKGROUND AND OBJECTIVE: Previous studies have demonstrated that our recombinant bacille Calmette-Guerin (rBCG), which expresses Der p2 in house dust mite (Der p2 rBCG) suppresses asthmatic airway inflammation by regulating the phenotype and function of dendritic cells (DC) and reprogramming T helper (Th) 0 cell differentiation into different T cell (Th1/Th2/Treg) subtypes. However, the exact role of Der p2 rBCG in reprogramming Th17 differentiation and the relevant mechanisms are not known. The aim of this study was to examine whether Der p2 rBCG-mediated inhibition of allergic airway inflammation is mediated by regulating Th17 differentiation in a murine asthma model. METHODS: Primary mouse bone marrow-derived dendritic cells (BMDC) were infected with Der p2 rBCG and adoptively transferred to Der p2-intranasally sensitized mice. The role of Der p2 rBCG-BMDC on the regulation of airway inflammation and Th17 cell differentiation was assessed. RESULTS: Adoptive transfer of Der p2 rBCG-BMDC suppressed airway inflammation and mucin secretion. Der p2 rBCG-BMDC inhibited excessive Th17 immune responses but not BCG-BMDC. Furthermore, Der p2 rBCG decreased jagged-2 and increased delta-like-4 expressions on BMDC to a greater extent than BCG. CONCLUSIONS: These findings suggest that DC plays a key role in Der p2 rBCG-induced immunoregulation. Der p2 rBCG also displayed a potent inhibitory effect on Th17 differentiation, and these findings increase our understanding of the cellular basis of Der p2 BCG-mediated inhibition of asthma.

Notch signaling downregulates MUC5AC expression in airway epithelial cells through Hes1-dependent mechanisms.

BACKGROUND: Mucus overproduction is one of the major pathological features of asthma, and MUC5AC is the major mucin component of airway mucus. However, whether Notch signaling is implicated in the regulation of MUC5AC expression in airway secretary cells is still undetermined. OBJECTIVE: The aim of this study is to examine whether Notch signaling can regulate MUC5AC expression and explore the molecular mechanisms. METHODS: Mouse mtCC1-2 cells and human NCI-H292 cells were transfected with NIC, and MUC5AC expression was examined. Using gene reporter assays, site-directed mutagenesis, and ChIP assays, the activity of both mouse and human MUC5AC promoter was analyzed. RESULTS: Notch signaling regulated MUC5AC expression both in mouse mtCC1-2 cells and in human NCI-H292 cells. Several Hes-binding site N-boxes were identified in the 5' region of both mouse and human MUC5AC promoters. Overexpression of NIC resulted in activation of the MUC5AC promoter. Site-directed mutagenesis report assays revealed that Hes proteins might repress both mouse and human MUC5AC promoter activity. Furthermore, ChIP assays confirmed that Hes1 binds to the MUC5AC promoter both in mouse mtCC1-2 cells and in human NCI-H292 cells. CONCLUSIONS: Notch signaling can directly downregulate MUC5AC promoter activity through Hes1-dependent mechanisms, which may be identified as possible targets for pharmacotherapy of airway mucus hypersecretion in asthma.

Der p 2 recombinant bacille Calmette-Guérin targets dendritic cells to inhibit allergic airway inflammation in a mouse model of asthma.

BACKGROUND: Previous studies showed that a recombinant bacille Calmette-Guérin (rBCG) which expressed the Der p 2 of house dust mites (Der p 2 rBCG) could suppress asthmatic airway inflammation. There are two possible mechanisms: (1) Der p 2 rBCG elicits immune deviation from Th2 to Th1, and (2) Der p 2 rBCG induces antigen-specific regulatory T cells. However, the role of dendritic cell (DC) Der p 2 rBCG in this protective effect and in reprogramming T-cell commitment still needs to be studied. OBJECTIVES: The aim of this study was to determine whether DCs play a central role in the Der p 2 rBCG-mediated inhibition of allergic airway inflammation. METHODS: DCs were collected from Der p 2 rBCG-immunized mice (Der p 2 rBCG-DCs) and adoptively transferred to Der p 2-sensitized mice. The effects of DCs on airway inflammation and immune regulation were analyzed. RESULTS: Adoptive transfer of DCs from Der p 2 rBCG-immunized mice suppressed asthmatic responses, including airway inflammation, mucin secretion and airway responsiveness. Der p 2 rBCG-DCs could effectively inhibit excessive Th2 immune responses and induced a subtype of CD4+CD25+Foxp3+ anti-specific regulatory T cells in this asthma model. Furthermore, Der p 2 rBCG immunization recruited more plasmacytoid DCs in abdominal draining lymph nodes. CONCLUSIONS: These findings suggest that DCs played a key role in Der p 2 rBCG-induced immunoregulation. Compared with BCG, Der p 2 rBCG displayed a more potent inhibitory effect on asthma responses, which may be related to the increase in plasmacytoid DC recruitment. These results improve our understanding of the cellular basis of Der p 2 BCG-mediated inhibition of asthma.

The role of bone marrow-derived adult stem cells in a transgenic mouse model of allergic asthma.

BACKGROUND: Asthmatic airway remodeling is an abnormal injury/repair process of the small airways caused by chronic inflammation in which the quantities of multiple cells increase dramatically. However, the origin of these proliferative cells is still undetermined. OBJECTIVE: The aim of this study was to examine whether bone marrow (BM)-derived adult stem cells are responsible for the proliferative cells in asthmatic airway remodeling. METHODS: Adult mice were durably engrafted with BM isolated from green fluorescent protein (GFP) transgenic mice. Using GFP BM chimera mice, an ovalbumin (OVA)-induced chronic asthma mouse model was established. The distribution of BM-derived GFP+ cells in the lungs of chronic asthma mice was detected by fluorescence microscopy. The phenotype of BM-derived GFP+ cells in the lung tissues of chronic asthma mice was analyzed by flow cytometry. RESULTS: BM chimera mice were successfully generated, with no detectable radioactive inflammation observed. Using BM chimera mice, we established a mouse model of chronic asthma characterized by a significant increase in the thickness of the airway subepithelial basement membrane and smooth muscle layers. OVA treatment caused many GFP+ cells to appear at sites of small airway inflammation. The extravascular localization of some GFP+ cells and their morphology were not consistent with leukocytes. Flow cytometric analysis of lung cells revealed a significant increase in type I collagen (Col I)+GFP+ cells and α-smooth muscle actin (α-SMA)+GFP+ cells in OVA-treated GFP BM chimera mice. CONCLUSIONS: Considerable numbers of Col I- and α-SMA-producing cells originated from BM in the lung tissues of mice with OVA-induced chronic asthma.

Suppression of allergic airway inflammation in a mouse model by Der p2 recombined BCG.

Allergic asthma is a chronic inflammatory disease mediated by T helper (Th)2 cell immune responses. Currently, immunotherapies based on both immune deviation and immune suppression, including the development of recombinant mycobacteria as immunoregulatory vaccines, are attractive treatment strategies for asthma. In our previous studies, we created a genetically recombinant form of bacille Calmette-Guerin (rBCG) that expressed Der p2 of house dust mites and established that it induced a shift from a Th2 response to a Th1 response in naive mice. However, it is unclear whether rBCG could suppress allergic airway inflammation in a mouse model. In this article we report that rBCG dramatically inhibited airway inflammation, eosinophilia, mucus production and mast cell degranulation in allergic mice. Analysis of interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) levels in bronchoalveolar lavage fluid (BALF) and lung tissue revealed that the suppression was associated with a shift from a Th2 response to a Th1 response. At the same time, rBCG induced a CD4(+) CD25(+) Foxp3(+) T-cell subtype that could suppress the proliferation of Th2 effector cells in vitro in an antigen-specific manner. Moreover, suppression of CD4(+) CD25(+) T cells could be adoptively transferred. Thus, our results demonstrate that rBCG induces both generic and specific immune responses. The generic immune response is associated with a shift from a Th2 to a Th1 cytokine response, whereas the specific immune response against Der p2 appears to be related to the expansion of transforming growth factor-beta (TGF-beta)-producing CD4(+) CD25(+) Foxp3(+) regulatory T cells. rBCG can suppress asthmatic airway inflammation through both immune deviation and immune suppression and may be a feasible, efficient immunotherapy for asthma.

Notch signaling regulates the FOXP3 promoter through RBP-J- and Hes1-dependent mechanisms.

Evidence has shown that Notch signaling modulates CD4(+)CD25(+) regulatory T-cells (Tregs). As transcription factor Foxp3 acts as a master molecule governing the development and function of Tregs, we investigated whether Notch signaling might directly regulate Foxp3 expression. Here, we provide evidence that Notch signaling can modulate the FOXP3 promoter through RBP-J- and Hes1-dependent mechanisms. A conserved RBP-J-binding site and N-box sites were identified within the FOXP3 promoter. We show that the Notch intracellular domain (NIC), the active form of Notch receptors, activates a reporter driven by the FOXP3 promoter. Dissection of the FOXP3 promoter revealed bipartite effects of the RBP-J-binding site and the N-boxes: the RBP-J-binding site positively, while the N-boxes negatively regulated the FOXP3 promoter activity. Moreover, in freshly isolated Tregs, NIC-RBP-J complex is bound to the FOXP3 promoter in Tregs. Our results suggest that Notch signaling might be involved in the development and function of Tregs through regulating Foxp3 expression.

Therapeutic efficacy of a co-blockade of IL-13 and IL-25 on airway inflammation and remodeling in a mouse model of asthma.

Repeated airway inflammation and unremitting remodeling provoke irreversible pulmonary dysfunction and resistance to current drugs in patients with chronic bronchial asthma. Interleukin (IL)-13 and IL-25 play an important role in airway inflammation and remodeling in asthma. We aimed to investigate whether co-inhibiting IL-13 and IL-25 can effectively down-regulate allergen-induced airway inflammation and remodeling in mice. Mice with asthma induced by chronic exposure to ovalbumin (OVA) were given soluble IL-13 receptor α2 (sIL-13R) or soluble IL-25 receptor (sIL-25R) protein alone and in combination to neutralize the bioactivity of IL-13 and IL-25, and relevant airway inflammation and remodeling experiments were performed. We found that the co-blockade of IL-13 and IL-25 with sIL-13R and sIL-25R was more effective than either agent alone at decreasing inflammatory cell infiltration, airway hyperresponsiveness (AhR) and airway remodeling including mucus production, extracellular collagen deposition, smooth muscle cell hyperplasia and angiogenesis in mice exposed to OVA. These results suggest that the combined inhibition of IL-13 and IL-25 may provide a novel therapeutic strategy for asthma, especially for patients who are resistant to current treatments.

KyoT2 downregulates airway remodeling in asthma.

The typical pathological features of asthma are airway remodeling and airway hyperresponsiveness (AHR). KyoT2, a negative modulator of Notch signaling, has been linked to asthma in several previous studies. However, whether KyoT2 is involved in the regulation of airway remodeling or the modulation of airway resistance in asthma is unclear. In this study, we aimed to evaluate the therapeutic potential of KyoT2 in preventing asthma-associated airway remodeling and AHR. BALB/c mice were used to generate a mouse model of asthma. Additionally, the expression of Hes1 and Notch1 in airway was analyzed using Immunofluorescence examination. The asthmatic mice were intranasally administered adenovirus expressing KyoT2 and were compared to control groups. Furthermore, subepithelial fibrosis and other airway remodeling features were analyzed using hematoxylin and eosin staining, Van Gieson's staining and Masson's trichrome staining. AHR was also evaluated. This study revealed that KyoT2 downregulated the expression of Hes1, repressed airway remodeling, and alleviated AHR in asthmatic mice. It is reasonable to assume that KyoT2 downregulates airway remodeling and resistance in asthmatic mice through a Hes1-dependent mechanism. Therefore, KyoT2 is a potential clinical treatment strategy for asthma.

Notch signaling regulates col1α1 and col1α2 expression in airway fibroblasts.

Subepithelial fibrosis is one of the common pathological features of asthmatic airway remodeling. During subepithelial fibrosis, type I collagen becomes the most abundant extracellular protein component. Studies have shown that Notch signaling participates in the progression of fibrosis; however, whether Notch signaling is involved in regulating type I collagen expression in airway fibroblasts remains unclear. The aim of the present study was to examine whether Notch signaling can regulate type I collagen expression in airway fibroblasts and to explore the underlying molecular mechanisms. Here, the expression of Notch signaling components was examined in mouse L929 cells and human MRC-5 cells. After upregulating or downregulating Notch signaling in these cell lines, col1α1 and col1α2 expression was examined. Using gene reporter assays, site-directed mutagenesis, and ChIP assays, the role of Hes1 binding sites in both the mouse and human COL1A1 and COL1A2 promoters was investigated. This study revealed that Notch signaling-related molecules (including Notch1, Hes1, and others) are expressed in L929 and MRC-5 cells and that Notch signaling regulates the expression of col1α1 and col1α2 in both cell lines. Additionally, over-expression of the Notch intracellular domain resulted in activation of the COL1A1 and COL1A2 promoters, and site-directed mutagenesis reporter assays revealed that Hes1 proteins might augment both mouse and human COL1A1 and COL1A2 promoter activity. Furthermore, ChIP assays confirmed that Hes1 binds to the COL1A1 and COL1A2 promoters in both L929 and MRC-5 cells. Therefore, it is reasonable to assume that Notch signaling can directly upregulate COL1A1 and COL1A2 promoter activity through a Hes1-dependent mechanism, which could serve as a possible target for pharmacotherapy of airway subepithelial fibrosis.

Suppression of allergic airway inflammation in a mouse model of asthma by exogenous mesenchymal stem cells.

Mesenchymal stem cells (MSCs) have significant immunomodulatory effects in the development of acute lung inflammation and fibrosis. However, it is still unclear as to whether MSCs could attenuate allergic airway inflammation in a mouse model of asthma. We firstly investigated whether exogenous MSCs can relocate to lung tissues in asthmatic mice and analyzed the chemotactic mechanism. Then, we evaluated the in vivo immunomodulatory effect of exogenous MSCs in asthma. MSCs (2 × 10(6)) were administered through the tail vein to mice one day before the first airway challenge. Migration of MSCs was evaluated by flow cytometry. The immunomodulatory effect of MSCs was evaluated by cell counting in bronchoalveolar lavage fluid (BALF), histology, mast cell degranulation, airway hyperreactivity and cytokine profile in BALF. Exogenous MSCs can migrate to sites of inflammation in asthmatic mice through a stromal cell-derived factor-1α/CXCR4-dependent mechanism. MSCs can protect mice against a range of allergic airway inflammatory pathologies, including the infiltration of inflammatory cells, mast cell degranulation and airway hyperreactivity partly via shifting to a T-helper 1 (Th1) from a Th2 immune response to allergens. So, immunotherapy based on MSCs may be a feasible, efficient therapy for asthma.