The Role of Macrophages in Pulmonary Regeneration using a Bioengineered Whole Lung Tissue Model

项目摘要/摘要 纤维性肺重塑是许多肺部疾病的标志，包括严重的流感感染、特发性 肺纤维化和新冠肺炎。虽然基底样上皮细胞已被确定为这一过程的中心参与者 异常修复反应，几乎没有做过研究其他细胞的行为和相互作用。 病变组织，尤指肺泡巨噬细胞。因此，这项研究的目标是利用一种仿生 工程化肺模型系统研究再生的基底样祖细胞之间的关系 和肺巨噬细胞。这种工程化的肺组织系统是基于细胞再繁殖和培养。 一种脱细胞的天然大鼠肺支架。该平台实现了受控良好的、类似于本地的组织环境 用于评估细胞间的相互作用，没有体内研究的系统性混杂因素。预计 巨噬细胞将显著影响和指导这些基底样细胞的上皮重塑，特别是在 与巨噬细胞的纤维化或抗纤维化激活状态有关。这项工作在以前发现的基础上进行了扩展 在含有基底样祖细胞的工程化肺培养中加入巨噬细胞显著改善了 组织结构和再生上皮细胞表型与无组织工程肺培养的比较 巨噬细胞。首先，通过支气管肺泡灌洗分离大鼠肺巨噬细胞，并 特色化的。将开发一种方案，在体外用化学方法刺激巨噬细胞形成一种疾病样细胞 炎症状态，或修复性抗纤维化状态。接下来，不同激活状态的巨噬细胞将被 将其引入再生基底细胞的气液界面培养，以评价上皮巨噬细胞 孤立的相互作用。最后，激活的巨噬细胞将被引入工程肺培养物中，其中包含 再生的基底细胞、成纤维细胞和内皮细胞，概括了基本的天然细胞群落。 工程肺组织也将在不同条件下进行组织学和生物力学变化的评估。 作为不同的细胞信号模式，通过单细胞RNA测序进行评估。预计 炎性巨噬细胞有助于再生上皮的纤维化反应，而抗纤维化 巨噬细胞将通过基底样细胞促进功能更强的肺泡再生。这项研究的发现 研究将阐明肺巨噬细胞在该模型中调节肺修复和再生的作用 系统。此外，这项工作可能会为纤维化肺部疾病的治疗提供可能的途径。建议数 研究项目将由大卫·H·科赫整合癌症研究所的艾莉森·M·格里尼执行 在罗伯特·兰格博士的赞助下，麻省理工学院(MIT)的研究，以及 耶鲁大学鲁斯兰·梅德日托夫博士的共同赞助。兰格博士和梅德日托夫博士将指导艾莉森 她的研究和专业培训目标是发展新的研究和科学沟通技能，因此 她可能具备在顶尖研究机构获得终身教职的条件。

PROJECT SUMMARY/ABSTRACT Fibrotic lung remodeling is a hallmark of many lung diseases, including severe influenza infection, idiopathic pulmonary fibrosis, and COVID-19. While a basal-like epithelial cell has been identified as a central player to this aberrant repair response, little has been done to investigate the behavior and interactions of other cells in the diseased tissue, particularly alveolar macrophages. Therefore, the goal of this study is to leverage a biomimetic engineered lung model system to investigate the relationship between regenerating basal-like progenitor cells and pulmonary macrophages. This engineered lung tissue system is based on cellular repopulation and culture of a decellularized native rat lung scaffold. This platform enables a well-controlled, native-like tissue environment for the evaluation of cell-cell interactions, without the systemic confounders of in vivo studies. It is expected that macrophages will significantly influence and direct epithelial remodeling by these basal-like cells, particularly in relation to the fibrotic or anti-fibrotic activation state of the macrophages. This work expands on previous findings that adding macrophages to engineered lung cultures containing basal-like progenitor cells significantly improves tissue architecture and regenerative epithelial cell phenotype, compared to engineered lung cultures without macrophages. First, pulmonary macrophages will be isolated from rats by bronchoavleolar lavage and characterized. Protocols will be developed to chemically stimulate macrophages in vitro to a disease-like inflammatory state, or to a reparative anti-fibrotic state. Next, macrophages of different activation states will be introduced to air-liquid interface cultures of regenerative basal cells, to evaluate epithelial-macrophage interactions in isolation. Finally, activated macrophages will be introduced to engineered lung cultures containing regenerative basal cells, fibroblasts, and endothelium to recapitulate essential native cellular communities. Engineered lung tissues will be evaluated for histologic and biomechanic changes between conditions, as well as differential cell signaling patterns, as evaluated by single-cell RNA sequencing. It is expected that inflammatory macrophages will contribute to fibrotic response in regenerating epithelium, whereas anti-fibrotic macrophages will contribute to more functional alveolar regeneration by basal-like cells. The findings of this study will elucidate the role of pulmonary macrophages in governing lung repair and regeneration in this model system. Further, this work may suggest possible routes for the treatment of fibrotic lung diseases. The proposed research project will be executed by Allison M. Greaney at the David H. Koch Institute for Integrative Cancer Research at the Massachusetts Institute of Technology (MIT), under the Sponsorship of Dr. Robert Langer, and Co-Sponsorship of Dr. Ruslan Medzhitov at Yale University. Dr. Langer and Dr. Medzhitov will mentor Allison in her Research and Professional Training Goals to develop new research and scientific communication skills, so she may be well-equipped to secure a tenure-track faculty position at a top research institution.