Immuno-stromal axes regulate fibroblast heterogeneity in tissue fibrosis and regeneration

项目摘要 由于损伤、疾病或先天性缺陷引起的组织损伤仍然是治疗糖尿病的关键临床障碍。 人体健康基于再生医学的疗法是一种很有前途的策略，然而， 免疫反应和纤维化。通常，在组织修复中，炎症受到严格控制， 然后由活化的成纤维细胞重塑，其存在也受到严格调控。免疫调节是一种 有希望的策略，以避免纤维化的结果，但它不是免疫细胞直接参与组织 重塑和胶原蛋白生成。因此，了解免疫细胞如何协调基质细胞是至关重要的。 细胞以促进组织修复或纤维化。在纤维化中，有证据表明慢性免疫细胞浸润和 促炎性细胞因子伴随着成纤维细胞准备促进纤维化。这些成纤维细胞的特征是 通过异常的机械传导（即感觉“正常”/软如硬/疤痕）导致过度生产 ECM。我们现在知道，免疫细胞和成纤维细胞作为表型不同的亚群存在，包括 那些与纤维化有关的分子特征。炎症信号是否引起不同的成纤维细胞 纤维化中具有异常机械转导的亚群是未知的，并且是关键目标。理解 炎症和成纤维细胞异质性之间的关系对于预测再生性纤维化是至关重要的。 治疗的潜力。我假设纤维化过程中存在成纤维细胞亚群， 机械传导受炎症信号调节。在K99阶段， 应用，我将确定免疫和成纤维细胞亚群出现在组织再生， 纤维化利用非纤维化/再生微孔退火颗粒水凝胶（MAP凝胶）和纤维化 无孔本体水凝胶（NP凝胶）。我将确定这些细胞之间的机械传导差异， 通过用鉴定的标记物纯化它们并测量ECM产生、肌纤维母细胞 分化、整合素活化和粘着斑信号传导。我将识别出 成纤维细胞亚群特有的表型。我将通过确认已确定的 纤维化临床植入物中的成纤维细胞亚群和信号通路。我将继续我的科学和 专业发展，为独立阶段做准备，并在我的科学指导下， 咨询委员会，多样性咨询委员会和合作者。在独立阶段，我将 使用MAP和NP凝胶确定再生和纤维化微环境特有的介质。I determine 如果通过在成纤维细胞特异性IL-1 RI敲除中植入NP凝胶， 小鼠我还将使用偶联有以下的MAP凝胶来确定IL-1信号传导是否足以促进纤维化： IL-1 β我们提出了一个创新的假设，试图弥合炎症和破坏之间的差距。 纤维化疾病的机械传导特征。这一建议将使我具备科学和专业的 培训，这将使我能够启动一个成功的和多样化的研究计划，作为一个独立的调查员。

Project Summary Tissue damage due to injury, disease, or congenital defect continues to be a critical clinical obstacle in human health. Regenerative medicine-based therapies are a promising strategy, yet the critical determinants of success are the immune response and fibrosis. Typically, in tissue repair, inflammation is closely controlled before remodeling by activated fibroblasts, whose presence is also tightly regulated. Immunomodulation is a promising strategy to avoid fibrotic outcomes, yet it is not the immune cells that directly participate in tissue remodeling and collagen production. Therefore, it is critical to understand how immune cells coordinate stromal cells to promote tissue repair or fibrosis. In fibrosis, evidence suggests that chronic immune cell infiltration and proinflammatory cytokines accompany fibroblasts poised to promote fibrosis. Those fibroblasts are characterized by aberrant mechanotransduction (i.e. sensing “normal”/soft as stiff/scarred) leading to the overproduction of ECM. We now know that immune cells and fibroblasts exist as phenotypically distinct subpopulations, including those with a molecular signature tied to fibrosis. Whether inflammatory signals elicit distinct fibroblast subpopulations with aberrant mechanotransduction in fibrosis is unknown and is a key objective. Understanding the relationship between inflammation and fibroblast heterogeneity is essential to predict the regenerative potential of therapies. I hypothesize that fibroblast subpopulations exist during fibrosis with aberrant mechanotransduction that is regulated by inflammatory signaling. During the mentored K99 phase of this application, I will identify the immune and fibroblast subpopulations that emerge during tissue regeneration and fibrosis leveraging non-fibrotic/regenerative microporous annealed particle hydrogels (MAP gels) and fibrotic nonporous bulk hydrogels (NP gels). I will determine the mechanotransductive differences across those subpopulations by purifying them with identified markers and measuring ECM production, myofibroblastic differentiation, integrin activation, and focal adhesion signaling. I will identify signaling pathways necessary for the phenotypes unique to fibroblast subpopulations. I will validate these findings by confirming the identified fibroblast subpopulations and signaling pathways in fibrotic clinical implants. I will continue my scientific and professional development in preparation for the independent phase with consistent guidance from my scientific advisory committee, diversity advisory committee, and collaborators. During the independent phase, I will determine mediators unique to regenerative and fibrotic microenvironments using MAP and NP gels. I determine if IL-1 signaling in fibroblasts is necessary for fibrosis by implanting NP gels in fibroblast-specific IL-1RI knockout mice. I will also determine whether IL-1 signaling is sufficient to promote fibrosis using MAP gels conjugated with IL-1. We pose an innovative hypothesis that seeks to bridge the gap between inflammation and disrupted mechanotransduction distinctive of fibrotic disorders. This proposal will equip me with scientific and professional training that will enable me to launch a successful and diverse research program as an independent investigator.