Lineage Commitment of Mesenchymal Stem Cell in Allergen-induced Airway Remodeling

描述(申请人提供)：哮喘是一种常见的慢性疾病，影响世界各地的许多儿童和成人。预防和治疗哮喘的一个主要障碍是其病因多样，以及我们对其生物学机制的了解不足。最近的研究改变了我们对哮喘作为一种纯粹的炎症性疾病的理解，变成了一种炎症和结构成分同等参与的疾病。过敏原在早期阶段引起的上皮损伤允许环境过敏原进入呼吸道组织，并可能导致慢性期气道重塑的发展。因此，早期针对受损上皮修复的治疗可以预防病理性气道重塑，改善哮喘控制。间充质干细胞(MSCs)是成体结缔组织祖细胞，具有多向分化潜能和潜在的旁分泌免疫调节特性。过敏原攻击后，肺内MSCs显著增加，可能参与了气道修复/重塑。转化生长因子1在干细胞/祖细胞的募集中起着重要作用，用于组织修复、再生和各种器官的重塑。我们之前的研究已经提供了初步证据，表明在过敏原诱导的哮喘模型中，MSCs和活跃的转化生长因子？1信号在呼吸道中增加。此外，蟑螂提取物的条件培养液(ECM)可诱导MSCs迁移，而转化生长因子1中和抗体可拮抗这种迁移。最近的体外研究表明，在有转化生长因子β1存在的情况下，间充质干细胞可以分化为上皮细胞。因此，关键阶段是评价转化生长因子β信号在哮喘中对间充质干细胞功能的影响。假设：活性转化生长因子1是一种过敏原激活的内源性信使，它将骨髓来源的MSCs招募到受损的呼吸道，在早期分化为上皮细胞修复受损的上皮细胞，或者在哮喘慢性期分化为纤维母细胞/肌成纤维细胞，参与纤维化的气道重塑。目的1提出实验以确定活性转化生长因子1在哮喘患者间充质干细胞向肺内募集中的作用。我们将使用我们的基于气液界面(ALI)-ECM的细胞迁移系统来确定转化生长因子？1在MSCs迁移中的作用。我们将利用NES-GFP小鼠进一步检测内源性Nestin+MSCs在哮喘不同阶段过敏原激发的气道中的募集情况，然后使用转化生长因子1中和抗体或转化生长因子受体I型(T？RI)抑制剂检测转化生长因子β1在间质干细胞募集中的作用。[目的2]提出了追踪哮喘急性期和慢性期通过转化生长因子1信号在肺中招募的MSC的谱系承诺/分化的实验。我们已经建立了一种可诱导的MSC谱系追踪小鼠模型(Nestin-CreERT2；rosa26-EGFP)，我们将使用该模型来跟踪急、慢性变应原诱导哮喘模型中招募的MSCs在气道中的谱系承诺。这将为了解转化生长因子-1信号和间充质干细胞在过敏性哮喘中的作用提供新的见解，并为新的治疗方法提供机会。

DESCRIPTION (provided by applicant): Asthma is a common chronic illness that affects many children and adults worldwide. A major obstacle to prevention and treatment of asthma has been its diverse etiologies and our inadequate understanding of the biological mechanisms. Recent studies have changed our understanding of asthma as a purely inflammatory disease to a disease in which both inflammatory and structural components are equally involved. Allergen caused epithelial damage in early stages allows environmental allergens access to the airway tissue and may lead to the development of airway remodeling in chronic stages. Therefore, therapies that target the repair of the damaged epithelium in early stages could prevent the pathological airway remodeling and improve asthma control. Mesenchymal stem cells (MSCs) are adult connective tissue progenitor cells with multi-lineage differentiation potential and poten paracrine immunomodulatory properties. MSCs are significantly increased in the lungs after allergen challenge and may participate in airway repair/remodeling. Transforming growth factor ¿1 (TGF¿1) plays an important role in the recruitment of stem/progenitor cells for tissue repair, regeneration, and remodeling in various organs. Our previous studies have provided initial evidence that MSCs and active TGF¿1 signaling are increased in airway in an allergen induced asthma model. Furthermore, conditioned medium (ECM) from cockroach extract challenged epithelium induces the migration of MSCs, while TGF¿1 neutralizing antibody antagonized this migration. Recent studies in vitro have shown that MSCs can differentiate into epithelial cells in the presence of TGF¿1. The stage is thus set to critically evaluate the functional effect on MSCs of TGF¿1signaling in asthma. HYPOTHESIS: Active TGF¿1 is an allergen-activated endogenous messenger that recruits bone marrow-derived MSCs to the injured airways, which differentiate into epithelial cells to repair the damaged epithelium in early stages or into fibroblasts/myofibroblasts contributing to fibrotic airway remodeling in chronic stage of asthma. Aim 1 proposes experiments to determine the role of active TGF¿1 in the recruitment of MSCs to the lung in asthma. We will determine the role of TGF¿1 in migration of MSCs using our Air-Liquid Interface (ALI)-ECM-based cell migration system. We will further examine the recruitment of endogenous nestin+MSCs to the allergen-challenged airways during different stages of asthma using Nes-GFP mice, followed by the examination of the role of TGF¿1 in the recruitment of MSCs using a TGF¿1 neutralizing antibody or TGF-¿ receptor type I (T¿RI) inhibitor. [Aim 2 proposes experiments to track the lineage commitment/differentiation of MSC recruited in lungs through TGF¿1 signaling during acute and chronic stages of asthma]. We have established an inducible MSC lineage tracing mouse model (nestin-CreERT2; ROSA26-EGFP) that we will use to track the lineage commitment of recruited MSCs in airway of acute and chronic allergen-induced models of asthma. This will provide novel insights into the role of TGF-¿1 signaling and MSCs in allergic asthma and offer an opportunity for novel therapies.