Pulmonary Organoid Models Demonstrate Compositionally Driven Epithelial Plasticity and Immune Polarization

Aberrant epithelial regeneration and immune remodeling are hallmarks of chronic lung diseases such as idiopathic pulmonary fibrosis (IPF), COPD, and post-viral syndromes. Yet how cellular context shapes these trajectories remains unresolved. We present a tunable, primary rat-derived lung organoid model that systematically varies immune, epithelial, and mesenchymal inputs to reveal how composition alone dictates epithelial plasticity and macrophage polarization. Across conditions, we observed the spontaneous emergence of disease-relevant transitional cell states, including Sox9⁺ stressed progenitors, RAS/AT0-like intermediates, and hillock-like cells, alongside distinct macrophage activation profiles. In mesenchyme-rich contexts, epithelial, immune, and mesenchymal crosstalk appeared to reinforce inflammatory signaling and stabilize transitional persistence, while immune-dominant inputs favored ATI-like repair and squamous remodeling. Notably, hillock-like cells displayed context-specific polarization and expressed immune-regulatory genes, suggesting a role as epithelial orchestrators that help calibrate inflammatory response during regeneration. Connectomic analysis revealed that regenerative outcomes were associated with dynamic multicellular signaling networks that integrate stress sensing, immune coordination, and epithelial fate. This platform provides a tractable system for modeling context-specific repair and regenerative mechanisms and could inform therapeutic strategies aimed at redirecting epithelial fate in chronic lung disease.上皮再生异常与免疫重塑是特发性肺纤维化（idiopathic pulmonary fibrosis, IPF）、慢性阻塞性肺疾病（chronic obstructive pulmonary disease, COPD）以及病毒感染后综合征等慢性肺部疾病的典型特征。然而，细胞微环境如何调控这些病理进程的发展轨迹仍未明确。本研究构建了一种可调控的原代大鼠源肺类器官模型，通过系统性改变免疫、上皮及间充质组分，以探究仅由组分构成即可决定上皮可塑性与巨噬细胞极化的机制。在不同培养条件下，我们观察到与疾病相关的过渡细胞状态自发出现，包括Sox9阳性应激祖细胞、RAS/AT0样中间细胞以及丘状细胞（hillock-like cells），同时伴随特征各异的巨噬细胞激活谱型。在富含间充质的微环境中，上皮、免疫与间充质细胞间的串扰会增强炎症信号通路并维持过渡细胞状态的持续存在；而以免疫组分为主的微环境则更倾向于ATI样修复与鳞状上皮重塑。值得注意的是，丘状细胞表现出微环境依赖性的极化特性，并表达免疫调控基因，提示其作为上皮调控因子，在再生过程中协助校准炎症应答。连接组学分析显示，再生结局与整合了应激感知、免疫协调及上皮细胞命运调控的动态多细胞信号网络密切相关。该平台为模拟微环境特异性的修复与再生机制提供了一个可操作的研究体系，有望为靶向重塑慢性肺部疾病中上皮细胞命运的治疗策略提供理论依据。