Bulk RNAseq analysis of Cx3cr1+ macrophages with and without IL4Ra deletion during bleomycin-induced lung fibrosis.. Bulk RNAseq analysis of Cx3cr1+ macrophages with and without IL4Ra deletion during bleomycin-induced lung fibrosis.

Wound healing in response to acute injury is mediated by the coordinated and transient activation of parenchymal, stromal, and immune cells that resolves to homeostasis. Environmental, genetic, and epigenetic factors associated with inflammation and aging can lead to persistent activation of the microenvironment and fibrosis. Here, we identify opposing roles of IL-4 cytokine signaling in interstitial macrophages and type II alveolar epithelial cells (ATIIs). We show that IL4Ra signaling in macrophages promotes regeneration of the alveolar epithelium after bleomycin-induced lung injury. Using organoids and mouse models, we show that IL-4 directly acts on a subset of ATIIs to induce the expression of the transcription factor SOX9 and reprograms them toward a progenitor-like state with both airway and alveolar lineage potential. In the contexts of aging and bleomycin-induced lung injury, this leads to aberrant epithelial cell differentiation and bronchiolization, consistent with cellular and histological changes observed in interstitial lung disease. Overall design: When culturing mouse alveolarspheres, we will seed mouse ATIIs from young and old mice, and grow them using serum-free and feeder-free system. On day 9, cells were infected with either AAV9_Cre or AAV9_Sox9Cre (to overexpress Sox9 together with Cre recombinase. Then we switched the culture medium to ATII differentiation medium to induce ATI differentiation for 4 days. We collected tdTomato+ cells by FACS and extracted nuclei for sc-multiome analysis.

急性损伤后的伤口愈合，由实质细胞、基质细胞与免疫细胞的协同瞬时激活所介导，最终恢复至机体稳态。
与炎症及衰老相关的环境、遗传与表观遗传因素，可导致组织微环境持续激活并引发纤维化。
本研究中，我们揭示了白细胞介素-4（IL-4）细胞因子信号在间质巨噬细胞与II型肺泡上皮细胞（type II alveolar epithelial cells，ATIIs）中的对立调控功能。
我们证实，巨噬细胞内的IL-4受体α链（IL4Rα）信号可促进博莱霉素（bleomycin）诱导的肺损伤后肺泡上皮再生。
借助类器官（organoids）与小鼠疾病模型，我们证实IL-4可直接作用于ATIIs的特定亚群，诱导转录因子SOX9的表达，并将其重编程为兼具气道与肺泡谱系潜能的祖细胞样状态。
在衰老与博莱霉素诱导的肺损伤情境下，这一过程会引发上皮细胞异常分化与细支气管化生，与间质性肺疾病（interstitial lung disease）中观察到的细胞及组织学改变高度一致。
整体实验设计：
在培养小鼠肺泡球时，我们将从年轻与老年小鼠体内分离并接种ATIIs，采用无血清、无饲养层培养体系进行扩增培养。于第9天，分别用AAV9_Cre或AAV9_Sox9Cre（用于同时过表达Sox9与Cre重组酶）感染细胞。随后将培养基更换为ATII分化培养基，诱导I型肺泡上皮细胞分化4天。我们通过荧光激活细胞分选术（FACS）收集tdTomato阳性细胞，提取细胞核用于单细胞多组学（sc-multiome）分析。