Damage-Associated Molecular Patterns Driving Fibrosis Progression in Scleroderma

2018年6月3日 摘要 多个器官的同步纤维化是系统性硬化症(SSC)的定义标志，但其 发病机制仍然知之甚少，迫切需要发现“可用药”的靶点。 模式识别受体Toll样受体4(TLR4)是一种重要的天然免疫调节因子。 在免疫细胞和基质细胞上均有表达，并可被内源性配体激活，称为 “损害相关的分子模式”(DAMPS)。从我们的出版和初步工作 实验室研究表明，TLR4及其内源性配体Tenascin-C可能在 SSc的多脏器纤维化。值得注意的是，Tenascin-C本身引起包括ECM在内的核心纤维化反应 成纤维细胞和三维人体皮肤器官模型中的产生和基质硬化。TLR4活动是 受泛素编辑酶A20调控，A20是SSC的主要风险基因。然而， 与SSC相关的A20变异体与发病机制的联系机制尚不清楚。我们演示了 在SSC皮肤活检中，A20的表达减少。令人惊讶的是，我们发现A20抑制了核心 纤维化反应，A20单倍体不足的小鼠表现出明显的非 炎症性皮肤纤维化。我们假设SSC中纤维化的持续性可以用以下方式解释 激活的TLR4信号，由tenascin-C和其他阻滞剂触发，并长期维持 由于A20功能受损。促纤维化TLR4途径活性增强的细胞类型，以及 纤维前病变的整体和特定的结构域仍不清楚。此外，临床上的 SSC中减少的A20与A20的细胞类型特异性调节和抗纤维化活性的相关性，以及 A20减少与SSC发病机制有关的机制从未被研究过。致信地址 这些关键的差距，目标1将决定细胞类型和刺激的特定角色和机制 两种不同实验诱导的多器官纤维化模型中的DAMP-TLR4信号 关键的Tenascin-C结构域并确定更多的DAMP作为潜在的SSC生物标志物和治疗 靶点：目标2将确定SSC纵向队列中A20表达的临床相关性 患者；并使用新的A20缺失和A20来确定A20在纤维化过程中的不同功能 人源化BAC转基因小鼠。目标3将确定TLR4的细胞来源和功能 SSc患者和对照组皮肤和肺中信号通路的活性。利用人类疾病 来自西北大学和耶鲁大学硬皮病项目的样本，结合体外 体内疾病模型和最先进的技术，包括全面的母体 对于多个组织的分析和无偏单细胞RNA测序，我们的研究团队已经做好准备 在理解SSC中的TLR4信号方面取得显著进展。这些信息将依次 指导发现新的生物标志物和治疗方法。

June 3 2018 ABSTRACT Synchronous fibrosis in multiple organs is the defining hallmark of systemic sclerosis (SSc), but its pathogenesis remains poorly understood, and there is an urgent need to discover “druggable” targets. The pattern recognition receptor Toll-like receptor 4 (TLR4), a vital mediator of innate immunity, is expressed on both immune and stromal cells, and can be activated by endogenous ligands called “damage-associated molecular patterns” (DAMPs). Published and preliminary work from our laboratories show that TLR4 and its endogenous ligand tenascin-C are likely to play important roles in multi-organ fibrosis in SSc. Notably, tenascin-C itself elicits core fibrotic responses including ECM production and matrix stiffening in fibroblasts and 3D human skin organoid models. TLR4 activity is regulated by the ubiquitin-editing enzyme A20, which is a major risk gene for SSc. However, the mechanism linking SSc-associated A20 variants and pathogenesis are unknown. We demonstrated that A20 expression is reduced in SSc skin biopsies. Surprisingly, we found that A20 inhibited core fibrotic responses, and mice that are haploinsufficient for A20 showed markedly aggravated non- inflammatory skin fibrosis. We hypothesize that persistence of fibrosis in SSc could be explained by activated TLR4 signaling that is triggered by tenascin-C and other DAMPs, and chronically sustained by impaired A20 function. The cell types with increased profibrotic TLR4 pathway activity, and the ensemble of profibrotic DAMPs and specific domains, remain unknown. Moreover, the clinical correlates of reduced A20 in SSc, cell type-specific regulation and anti-fibrotic activity of A20, and the mechanisms linking reduced A20 and SSc pathogenesis, have never been investigated. To address these critical gaps, Aim 1 will determine cell type- and stimulus-specific roles and mechanisms of DAMP-TLR4 signaling in two separate models of experimentally-induced multi-organ fibrosis; map key tenascin-C domains and identify additional DAMPs as potential SSc biomarkers and therapeutic targets; Aim 2 will define the clinical correlates of A20 expression in a longitudinal cohort of SSc patients; and define distinct functions of A20 in the fibrotic process using novel A20-deficient and A20 humanized BAC transgenic mice. Aim 3 will determine the cellular sources and function of TLR4 signaling pathway activity in skin and lung from SSc patients and controls. Employing human disease samples from the established Northwestern and Yale Scleroderma Programs, combined with in vitro and in vivo disease models and state-of-the-art technologies including comprehensive matrisome analysis and unbiased single cell RNA sequencing of multiple tissue, our investigative team is poised to generate notable advances in understanding TLR4 signaling in SSc. The information in turn will guide discovery of novel biomarkers and therapies.