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) 的明确标志，但其 发病机制仍知之甚少，迫切需要发现“可成药”的靶点。 模式识别受体 Toll 样受体 4 (TLR4) 是先天免疫的重要介质， 在免疫细胞和基质细胞上表达，并且可以被称为内源性配体的激活 “损伤相关分子模式”（DAMP）。我们已发表的和初步的工作 实验室表明 TLR4 及其内源性配体 tenascin-C 可能在 SSc 中的多器官纤维化。值得注意的是，生腱蛋白-C 本身可引发核心纤维化反应，包括 ECM 成纤维细胞和 3D 人体皮肤类器官模型中的生产和基质硬化。 TLR4 活性为 受泛素编辑酶 A20 调节，该酶是 SSc 的主要风险基因。然而， 连接 SSc 相关 A20 变异和发病机制的机制尚不清楚。我们展示了 SSc 皮肤活检中 A20 表达降低。令人惊讶的是，我们发现 A20 抑制核心 纤维化反应，单倍体 A20 不足的小鼠表现出明显加重的非纤维化反应。 炎症性皮肤纤维化。我们假设 SSc 中纤维化的持续存在可以通过以下方式解释： 激活的 TLR4 信号传导由生腱蛋白-C 和其他 DAMP 触发，并长期持续 A20 功能受损。促纤维化 TLR4 通路活性增加的细胞类型，以及 促纤维化 DAMP 和特定结构域的整体仍然未知。此外，临床 SSc 中 A20 减少、细胞类型特异性调节和 A20 抗纤维化活性的相关性，以及 A20 减少和 SSc 发病机制之间的联系机制从未被研究过。致地址 这些关键差距，目标 1 将确定细胞类型和刺激特异性的作用和机制 实验诱导的多器官纤维化的两个独立模型中的 DAMP-TLR4 信号传导；地图 关键的生腱蛋白-C 结构域，并确定其他 DAMP 作为潜在的 SSc 生物标志物和治疗药物 目标；目标 2 将定义 SSc 纵向队列中 A20 表达的临床相关性 病人；并使用新型 A20 缺陷型和 A20 定义 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 患者和对照者皮肤和肺部的信号通路活性。利用人类疾病 来自已建立的西北大学和耶鲁大学硬皮病项目的样本，结合体外 以及体内疾病模型和最先进的技术，包括综合基质体 对多个组织进行分析和公正的单细胞 RNA 测序，我们的研究团队已做好准备 在理解 SSc 中 TLR4 信号传导方面取得显着进展。该信息依次将 指导新型生物标志物和疗法的发现。

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.