Table_4_Senescence of alveolar epithelial cells impacts initiation and chronic phases of murine fibrosing interstitial lung disease.docx

Fibrosing interstitial lung disease (ILD) develops due to the impaired reparative processes following lung tissue damage. Cellular senescence has been reported to contribute to the progression of fibrosis. However, the mechanisms by which these senescent cells initiate and/or drive the progression of lung tissue fibrosis are not yet fully understood. We demonstrated that p21WAF1/CIP1- and p16INK4A-pathway-dependent senescence in type 2 alveolar epithelial cells (AEC2) were both involved in the initiation and progression of lung fibrosis in murine bleomycin (BLM)-induced ILD. p21WAF1/CIP1-senescent AEC2 emerged rapidly, as early as 1 day after the intratracheal instillation of BLM. Their number subsequently increased and persisted until the later fibrosis phase. Very few p16INK4A-senescent AEC2 emerged upon the instillation of BLM, and their increase was slower and milder than that of p21WAF1/CIP1+ AEC2. AEC2 enriched with senescent cells sorted from BLM-ILD lungs expressed senescence-associated secretory phenotype (SASP)-related genes, including Il6, Serpin1, Tnfa, Ccl2, Tgfb, and Pdgfa, at the initiation and chronic phases of fibrosis, exhibiting distinct expression patterns of magnitude that were dependent on the disease phase. Ly6C+ inflammatory monocytes increased in the lungs immediately after the instillation of BLM and interstitial macrophages increased from day 3. The expression of Acta2 and Col1a1 was upregulated as early as day 1, indicating the activation of fibroblasts. We speculated that IL-6, plasminogen activator inhibitor-1 (PAI-1), and TGF-β contributed to the accumulation of senescent cells during the progression of fibrosis in an autocrine and paracrine manner. In addition, CCL2, produced in large amounts by senescent AEC2, may have induced the infiltration of Ly6C+ inflammatory monocytes in the early phase, and TGF-β and PDGFa from senescent AEC2 may contribute to the activation of fibroblasts in the very early phases. Our study indicated that senescent AEC2 plays a role in the pathogenesis of fibrosing ILD throughout the course of the disease and provides insights into its pathogenesis, which may lead to the development of new therapeutic methods targeting senescent cells or SASP molecules.

纤维化间质性肺病（Fibrosing interstitial lung disease，简称ILD）之发生，源于肺组织损伤后修复过程的障碍。细胞衰老已被指出是纤维化进程加剧的诱因之一。然而，这些衰老细胞如何启动并/或驱动肺组织纤维化的具体机制，尚未得到充分阐释。本研究证实，在由博莱霉素（Bleomycin，简称BLM）诱导的鼠类ILD中，p21WAF1/CIP1-及p16INK4A通路依赖性衰老在2型肺泡上皮细胞（AEC2）中，既参与了纤维化的启动，也参与了其进展。p21WAF1/CIP1+衰老的AEC2细胞在BLM经气管内注入后迅速出现，其数量随时间增加并持续至纤维化后期。p16INK4A+衰老的AEC2细胞在BLM注入后出现较少，其数量的增加速度及程度均低于p21WAF1/CIP1+ AEC2细胞。从BLM-ILD肺中分离出的富含衰老细胞的AEC2在纤维化的启动和慢性阶段表达衰老相关分泌表型（SASP）相关基因，包括Il6、Serpin1、Tnfa、Ccl2、Tgfb和Pdgfa，其表达模式随疾病阶段而异，表现出显著差异。Ly6C+炎症单核细胞在BLM注入后立即增加，间质性巨噬细胞从第3天开始增加。Acta2和Col1a1的表达在注入BLM后第1天即上调，这表明成纤维细胞的激活。我们推测，IL-6、纤溶酶原激活物抑制剂-1（Plasminogen activator inhibitor-1，简称PAI-1）和TGF-β可能通过自分泌和旁分泌的方式，在纤维化进展过程中导致衰老细胞的积累。此外，由衰老AEC2大量产生的CCL2可能在早期阶段诱导Ly6C+炎症单核细胞的浸润，而来自衰老AEC2的TGF-β和PDGFa可能在非常早期阶段促进成纤维细胞的激活。本研究表明，衰老的AEC2在纤维化ILD的整个病程中发挥着作用，并对其发病机制提供了深刻的见解，这或许将引领针对衰老细胞或SASP分子的新型治疗方法的开发。