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 a...