Neuroimmune Molecules in Scleroderma Lung Fibrosis

 描述(申请人提供)：硬皮病(系统性硬化症，SSC)是一种以皮肤和内脏纤维化为特征的特发性自身免疫性疾病，许多患者受到间质性肺疾病(SSC-ILD)的影响，但缺乏特异、高效的治疗方法。SSC-ILD对免疫调节剂的反应，在某些情况下，骨髓移植提示白细胞参与了疾病的发病机制。对SSc-ILD患者肺组织的详细检查经常发现炎性细胞与正常和纤维化的细胞外基质(ECM)并列。因此，值得注意的是，纤维细胞，一组显示间充质特征的白细胞，在几项研究中显示出在SSC-ILD血液和/或肺中聚集增强。这些细胞的意义和调节它们在临床上出现的因素仍不清楚。我们实验室发表的和初步的工作表明，层粘连蛋白样神经免疫分子Netrin-1(NTN-1)在这种情况下很重要。NTN-1通过与其吸引力受体(在结直肠癌-1(DCC-1)中缺失)结合来刺激细胞吸引力，而细胞的排斥和侵袭是通过与其排斥性受体非协调5a(UNC5a)的相互作用来驱动的。我们使用一个基于去细胞人肺的新的翻译平台来探索这些途径与SSC-ILD的相关性，以证明产生胶原的白细胞的积累受人肺ECM的生化和机械方面的调节，并且当SSC-ILD PBMC暴露于纤维化硬皮病肺的可消化成分时，会产生更多的纤维细胞。在SSc-ILD中，CD14lo单核细胞上NTN-1的表达增加，它调节白细胞-基质相互作用和PBMC分泌转化生长因子-β-1。最后，在博莱霉素肺纤维化模型中进行的研究表明，NTN-1的部分缺乏可以改善纤维化的组织学和生化读数、转化生长因子-β1的产生和CD45Coliα1细胞的聚集，表明NTN-1的中和可能是在此背景下的治疗靶点。对这些区域的探索有可能促进对SSc肺受累的了解。因此，在目标1中，我们将使用标准的免疫定位技术和多参数质量细胞术来确定NTN-1、DCC-1和UNC5a在两个特征良好的SSC-ILD纵向队列患者的肺和血液中先天免疫细胞和获得性免疫细胞上的表达。在目标2中，我们使用中和抗体和病毒介导的过度表达策略，结合通过与正常和SSc-ILD肺基质结合而具有生物活性的机械可调聚合物网络来确定Netrin-1‘S对纤维细胞聚集的调控是由于受体介导的与细胞外基质成分的相互作用，还是来自于底层底物的蛋白质组成和硬度的组合影响。在目标3中，我们使用了uc5a和dcc-1零突变的小鼠，以及细胞特异性缺失的ntn-1和转化生长因子-β1的小鼠，以确定它们在两种不同的实验性诱导的肺纤维化模型中对纤维化和肺内纤维细胞聚集的贡献。

 DESCRIPTION (provided by applicant): Scleroderma (Systemic Sclerosis, SSc) is an idiopathic autoimmune disease characterized by cutaneous and visceral fibrosis in which many patients are affected by interstitial lung disease (SSc-ILD) which lacks specific, highly efficacious therapy. The response of SSc-ILD to immunomodulatory agents and in some cases bone marrow transplantation suggests involvement of leukocytes in disease pathogenesis. Detailed examination of lung tissue obtained from patients with SSc-ILD frequently reveals inflammatory cells juxtaposed with normal and fibrotic extracellular matrix (ECM). It is therefore notable that fibrocytes, a population of leukocytes displaying mesenchymal characteristics, have in several studies demonstrated enhanced accumulation in the SSc-ILD blood and/or lung. The significance of these cells and the factors regulating their appearance in this clinical context remains unknown. Published and preliminary work from our laboratory indicates the laminin-like neuroimmune molecule Netrin-1 (NTN-1) is important in this setting. NTN-1 stimulates cellular attraction via binding to its attractive receptor, Deleted in Colorectal Cancer-1 (DCC-1) while cellular repulsion and invasion is driven by interactions with its repulsive receptor, Uncoordinated-5a (UNC5a). We explored the relevance of these pathways to SSc-ILD using a novel translational platform based on decellularized human lungs to demonstrate that the accumulation of collagen-producing leukocytes is regulated by biochemical and mechanical aspects of the human lung ECM, and that SSc-ILD PBMCs produce more fibrocytes when exposed to the digestible components of the fibrotic Scleroderma lung. NTN-1 expression is increased on CD14lo monocytes in SSc-ILD where it regulates leukocyte-matrix interactions and TGF-β1 secretion by PBMCs. Last, studies performed in the bleomycin model of pulmonary fibrosis demonstrate that partial deficiency of NTN-1 ameliorates histologic and biochemical readouts of fibrosis, TGF-β1 production, and the accumulation of CD45+ColIα1+ cells, indicating that NTN-1 neutralization might be a therapeutic target in this context. Exploration of these areas has the potential to advance the understanding of lung involvement in SSc. Therefore, in Aim 1, we will use standard immunolocalization techniques and multiparametric mass cytometry to define the expression of NTN-1, DCC-1, and UNC5a on innate and adaptive immune cells in the lungs and blood of two well characterized longitudinal cohort of subjects with SSc-ILD. In Aim 2, we use neutralizing antibodies and virally mediated overexpression strategies combined with mechanically tunable polymer networks rendered bioactive through conjugation with normal and SSc-ILD lung matrix to determine whether Netrin-1's modulation of fibrocyte accumulation results from receptor mediated interactions with ECM components or from the combinatorial influence of the protein composition and stiffness of the underlying substrate. In Aim 3, we use mice with null mutations of UNC5a and DCC-1, as well as mice with cell specific deletion of NTN-1 and TGF-β1 to determine their contribution to fibrosis and intrapulmonary fibrocyte accumulation seen in two separate models of experimentally-induced lung fibrosis.