Functional Effect Of TGF β1 On Mesenchymal Stem Cells Mobilization In Cockroach Allergen-Induced Athma

BACKGROUNDAsthma is a serious chronic illness that affects an estimated 300 million people and has a severe public health impact and economic burden for the family and country. There were about 200-300 people died from asthma each year in United States. Asthma is a chronic airway inflammation disease, characterized by airway hyperresponsiveness, reversible airflow obstruction and the airway remodeling. The chronic airway inflammation is the core factor of the asthma. The common pathophysiology symptoms include more infiltrating cells such as Eosnophils and lymphocytes in the airway; the bronichial lining ciliated cell damaged or necrosis and the epithelial necrosis and falls off; the goblet cells increased and secrets a large number of mucus; smooth muscle hypertrophy, bronchial wall thickening and thickening of basement membrane. Recent study showed that the asthma is not only a purely inflammatory disease, but also is associated with structural remodeling of the airway characterized by airway epithelial damage, wall thickening, and subepithelial fibrosis. The remodeling of the airway is the principal factor that leads to airway hyperresponsiveness and the reversible airflow obstruction. The asthma is more prevalence in inner-city populations. It has been suggested that there is a strong link between allergen exposure and sensitization and development of asthma. In particular, exposure to cockroach allergen in early life can lead to allergic airway inflammation and increased risk of developing asthma. The National Cooperative Inner-City Asthma Study (NCICAS) showed that 60%-80% of children with asthma are sensitized to cockroach allergen. However, fundamental questions remain to be fully elucidated regarding the genesis of cockroach allergen extract (CRE) induced asthma.Bone marrow mesenchymal stem cells (BMSCs) are adult connective tissue progenitor cells with the ability of self-review and differentiation into tissues of mesodermal origin, such as adipocytes, chondroblasts, and osteoblasts. First identified and isolated from the bone marrow (BM), MSCs can now be expanded from a variety of other tissues including muscel, synovial membrane, lungs, liver, peripheral blood and cord blood and adipose tissue. In addition, with low immunogenicity and surface expression of all kinds of adhesion molecules and chemokines, MSCs can migrate to different tissues. Importantly, MSCs have been shown to modulate the immune responses of various immunocytes such as T cells, B cells and dendritic cells (DCs). Theoretically, MSCs can suppress lung inflammation and modulate the immune function. MSCs maybe can be a way to treat the asthma by modulating the inflammation cytokines secretion and the airway remodeling. However, the molecular mechanism underlying the effect of MSCs on asthma remains unclear.Transforming growth factor (31 (TGFβ1) is a multifunctional cytokine that plays a critical role in cell growth, differentiation, and immune regulation, and it has been considered a principal mediator of airway remodeling. TGFβ consists of three isoforms:TGFβ1, TGFβ2 and TGFP3, regulates a broad spectrum of biological processes. In these three isoforms, TGFβ1 has the highest proportion in human cells. Excessive TGFβ activation is a maor contributor to a variety of diseases including cancer, autoimmune disease, vascular diseases and progressive fibrosis in multiple organs. TGFPs are synthesized as latent form in a complex that is maintained in a sequestered state in extracellular matrix (ECM), thereby preventing the release of free TGFβ for its action, a process called latent TGFβ activation. Thus, the extracellular TGFβ activity is primarily regulated by the conversion of latent TGFβ to active TGFβ, and latent TGFP is considered as a molecular sensor that responds to specific signals by releasing active TGFβ. These signals are often perturbations of the ECM that are associated with phenomena such as mechanical stress, wound repair, tissue injury, and inflammation. TGF-β1 mediates signaling by binding to cell surface TGF-P type I and type II Ser/Thr kinase receptors, and leads to the phosphorylation of the receptor-regulated SMADs, i.e., SMAD2 and SMAD3. The phosphorylated SMAD2 and SMAD3 bind to SMAD4 and form a hetero-oligomer, and the hetero-oligomer then translocates into the nucleus, where it regulates the expression of TGF-β1 target genes. Recent studies have demonstrated that TGF-β1 is associated with airway remodeling. Specifically, increased active TGF-β1has been observed in airways from asthmatic patients and from experimental mice during allergic airway inflammation.In the present study, we have specifically focused on the TGF-β1 activation in the migration of MSCs, which maybe critical in modulating allergen-induced airway allergic inflammation. Firstly, we observed the lung inflammation and MSCs mobilization in our cockroach allergen induced asthma model. Secondly, isolate and culture the MSCs from bone marrow; thirdly, we observed the effect of TGF-β1 in MSCs migration in vivo and in vitro; lastly, we observed the effect of TGF-β1 on the function of MSCs in allergic inflammation.Materials and Methods1. In the present study, six to 8-wk-old female C57BL/6J mice and nestin-GPF-transgenic mice and C57BL/6J-GPF mice were used. All animals were maintained under specific pathogen-free conditions in the animal facility of the Johns Hopkins University School of Medicine. The experimental protocols were reviewed and approved by the Animal Care and Use Committee at Johns Hopkins University School of Medicine. And the project No is M013M308.2. The level of activated TGF-β1 in the Bronchoalveolar lavage fluid (BALF), serum and the condition medium was detected by ELISA. We also detected the expression of cytokines such as IL-4, IL-13, IL-17A and INFy in BALF by ELISA.3. The differential cell counts in BALF and the percent of Treg cells were measured by flow cytometry.4. H&E staining of lung tissue sections were performed to observe the lung inflammation; IHC staining for the nestin+and p-Smad2/3+cells. Immunofluorescent Staining of lung tissue sections were performed to observe the expression of p-Smad2/3 and GFP+ cells.5. Isolate and culture the BMSCs and identify the cells by inducing them to differentiation in to adipocytes, chondroblasts, and osteoblasts under different condition.6. The expression of p-Smad2/3 and Smad2/3 in MSCs after CRE treatment by western blot.7. The gene expression of cytokines and cell markers in M1 and M2 macrophage by RT-PCR.8. The transwell assay was use to observed the migration of MSCs in vitro.9. Data are expressed as the mean±SEM for each group. Statistical significance for normally distributed samples was assessed using an independent two-tailed Student t test for with ANOVA by using IBM SPSS statistics 22. Differences with P＜0.05 were considered statistically significant.Results1. The airway inflammation was increased in CRE induced asthma mouseThe mices were sensitized and challenged by CRE and PBS. Compared with PBS group, the total cell number increased and among them the number of eosinophils, macrophages, lymphocytes and graunocytes were also increased in CRE group. There are more infiltrating cells in the lung tissue and the cockroach allergen-specific IgE was increased in the CRE group.2. MSCs are accumulated in lung tissue of CRE-challenged miceIn order to investigate whether CRE can induce MSC migration to the lungs, we established a CRE induced asthmatic mouse model. Our histological anslysis demonstrated a high number of nestin+　cells in aireay epithelial cells and subepithelial inflammatory cells from mice after CRE challenge as compared with PBS group. No positive staining was seen for control IgG.3. TGFβ1 signaling is activated in lung tissue of allergic asthma and in CRE-treated MSCsOur recently studies have suggested that activated TGFβ1 released from the injured vessels controls mobilization and recruitment of MSCs to participate in tissue repair/remodeling. To investigate whether TGFβ1 signaling is involved in the migration of MSCs to lungs in asthma, we examined the levels of active TGFβ1 in blood and BALF in CRE-challenged mice. The concentrations of active TGFβ1 were significantly higher in both BALF and peripheral blood in mice after allergen challenge, when compared with control mice. We also detected p-Smad2/3 in the airways of the mice. As compared to those of saline-treated mice much higher numbers of phospho-Smad2/3+ (p-Smad2/3+) epithelial cells and subepithelial inflammatory cells were found in the airways of mice after CRE challenge. The results indicate that cockroach allergen induces the activation of TGFβ1 signaling in airways in asthma. To further examine whether cockroach allergen can induce the activation of TGFβ1 signaling in MSCs, we detected the expression of cockroach allergen induced TGFβ1 as well as p-Smad2/3 in MSCs. We found that MSCs can secrete a large amount of active TGFβ1 in response to cockroach allergen Furthermore, cockroach allergen can induce the increased activation of TGFβ1 signaling in MSCs as determined by western blotting and immunofluorescent staining.4. The morphology of BMSCsThe initial adherent spindle-shaped cells appear as individual cells on the third day in phase-contrast microscopy. Within 7-10 d, the culture becomes more confluent and reaches 80-90% confluence with 2 weeks. At this stage, the cultures typically exhibit two characteristics: first, plates may contain distinct colonies of fibroblastic cells that vary in size; and second may contain very small numbers of hematopoietic cells interspersed between or on the colonies.5. The differentiation of BMSCsIn order to get the more pure BMSCs we use the fluorescence activated cell sorting (FACS) get the Sca-1+ CD29+ CD11b- CD45" cells. BMSCs have the multilineage differentiation capacity of the cells:BMSCs were capable of osteoblasts, adipocytes and chondrocytes. In addition, The ability of BMSCs to differentiate into smooth muscle like cells was evaluated by the expression of α-smooth muscle actin when culture with appropriate media.6. TGFβ1 mediates MSC migration induced by human conditioned mediumTo further examine whether active TGFβ1 released from epithelium in response to environmental allergens can induce MSC migration, we performed a human epithelium conditioned medium (ECM)-based cell migration assay in which ECM collected by incubating human epithelium with or without CRE challenge was placed in the bottom chamber of a transwell and MSCs were placed in the upper chamber of the tranwell. Significantly greater numbers of migrated MSCs were observed in the group with ECM prepared from allergen challenged epithelial cells, compared to those from the resting cells. To examine whether TGFβ1 is one of the major factors in CRE-challenged ECM responsible for the increased migration of MSCs, we firstly measured the levels of active TGFβ1 in ECM. A significantly greater amount of active TGFβ1 was observed in ECM prepared by CRE challenged epithelial cells when compared to those from the resting cells. To further examine whether TGFβ1 in ECM is essential for MSC migration, we added TGFβ1 neutralizing antibodies in ECM for the migration assay. Interestingly, the migration of MSCs was almost abolished when TGFβ1 neutralizing antibody was used. The migration of MSCs was not affected by adding antibody against SDF-la/CXCL12, a chemoattractant that can induce the migration of many cell types. When different doses of TβR1 kinase-specific inhibitor SB50512 (0.5μM, 1.0μM, and 2.0μM) were added to ECM for the migration assay, the ECM induced migration was significantly inhibited in a concentration-dependent manner, suggesting that TGFβ1 signaling is a primary pathway that drives MSC migration. To validate the direct effect of TGFβ1 on MSC migration, we analyzed the migration of MSCs using different doses of recombinant TGFβ1 in the transwell assays. Significant MSC migration was seen at 5,10, and 20ng/ml TGFβ1 in a dose-dependent manner.7. TGFβ1 mediates the recruitment of MSCs to the lungs in asthmaWhile MSCs were increased in airway after CRE sensitization and challenge, it remains unclear whether these MSCs are bone marrow-derived or local in origin. We thus examined whether there was an increased recruitment of MSCs to the lung from peripheral blood or bone marrow. We use the Nes-GFP transgenic mice to tract the migration of MSCs in the asthma model. We alos examined whether TGFβ1 is essential for the recruitment of endogenous MSCs to the lungs in asthma. The mices were divided into 3 groups:PBS group, CRE group and CRE+TGFβAb group. Increased numbers of engrafted GFP+MSCs in the airways and in BAL were observed after the mice were treated with CRE. Importantly, the increased GFP+MSCs were significantly diminished in mice receiving TGFβ1 neutralizing antibody. To investigate whether MSCs can migrate from peripheral blood to the airway following CRE sensitization and challenge, a total of 1×106 sorted MSCs were then injected into CRE-challenged mice through the tail vein right before CRE challenge. The mices were divided into three groups: CRE+PBS group, CRE+MSCs group, CRE+ MSCs+ TGFβ1 group. Significantly greater numbers of GFP+ cells were observed in the lungs from these CRE+MSCs group, when compared with those in the lungs that received CRE alone. To examine whether TGFβ1 is required for GFP+ MSC recruitment, we systemically injected TGFβ1 neutralizing antibody on the day before MSC injection and CRE challenge. We found that the increased GFP+ MSCs in lungs of CRE-induced mice were significantly inhibited when the mice were pretreated with TGFβ1 neutralizing antibody.8. MSCs inhibits the CRE induced allergic inflammationAfter the mice injected the GFP+MSCs from the tail vain, we also investigate the effect of MSCs in the CRE induced allergic inflammation. Decreased recruitment of total inflammatory cells and neutrophils, eosinophils, macrophages, and lymphocytes were detected in BALF in CRE+ MSCs group. Moreover, dense peribronchial infiltrates were also decreased in the lung tissue by histological examination. In addition, the Th2 cytokines such IL-4, IL13 decreased and Thl cytokines such as IFN y decreased. TGFβ1 Ab treatment elevated total number of inflammatory cells in BAL, specifically eosinophils, increased the airway inflammation and increased the Th2 cytokines. The increased inflammation in lungs after treatment with TGFβ1 neutralizing antibody was correlated with decreased numbers of MSCs detected in the airway and in BAL.9. MSCs modulates the Treg cellsTo test whether MSCs suppresses inflammation through Treg, we examined the percentage of CD4+CD25+FoxP3 Treg cells in the lung hilar lymph nodes in TGFβ1 neutralizing antibody treated and untreated mice. MSCs upregulate the percentage of CD4+CD25+FoxP3 Treg cells when compared with CRE+PBS group. However, no significant difference was observed in the percentage of Treg cells between TGFβ1 neutralizing antibody treated and untreated groups.10. MSCs modulates macrophage polarizationAs we know, macrophage has the function of anti-inflammation and repairing the tissue. To investigate whether MSCs inhibit allergic inflammation by modulating the macrophage polarization, we detect the cytokines can cell markers in Ml and M2 macrophage phonotype by RT-PCR. MSCs increased the cell marker in M2 such as Arg-1, FIZZ1 and YM-1 and decreased M1 cytokines such as IL-1β, IL-6 and NOS2 when compared with CRE+PBS group. There was a trend to increase Ml cytokines and decrease M2 cell markers after injecting TGFβ1 neutralizing antibody.Conclusion1. We successfully established a cockroach allergen-induced asthmatic mouse model.2. MSCs are accumulated in lung tissue in CRE-challenged mice and and the TGFpi was activated after CRE treatment in MSCs and mice.3. We successfully Isolated, cultured and identified the BMSCs.4. TGFβ1 can modulate the MSCs migration in vitro and in vivo.5. MSCs limit allergic inflammation through decreasing the expression of Th2 cytokines, upregulating the percent of Treg cells and modulating the polarization of macrophage. The TGFβ1 signaling plays an important role in the process of suppressing inflammation.Taken together, our studies have demonstrated that TGFβ1 released from allergen-activated epithelium is a primary promigratory factor controlling the recruitment of MSCs to the lungs in asthma. Moreover, we demonstrated that MSCs can inhibit CRE-induced allergic inflammation. TGFβ1 may play a role in allergen-induced airway allergic inflammation. Importantly, these studies provide a basis for the clinical treatment of asthma.