Abstract Objective Benign tracheal stenosis (BTS) results from various etiologies and is characterized by persistent inflammation and aberrant tissue repair after airway injury. It is associated with substantial harm to patients and represents a pressing clinical challenge in need of effective solutions. However, the specific inflammatory cell subtypes involved and their underlying molecular mechanisms remain largely unclear. Secreted phosphoprotein 1 (SPP1, osteopontin) plays a critical role in regulating immune cell function and tissue remodeling. This study aimed to characterize the functional features and regulatory mechanisms of SPP1-high-expressing macrophages (Mac-SPP1hi) in BTS, and to identify potential therapeutic targets for immune intervention. Methods Single-cell RNA sequencing (scRNA-seq) was performed on tracheal granulation tissue from BTS patients and normal controls to delineate transcriptional changes in macrophage subpopulations. Multi-omics integration identified Galectin-1 (Gal-1) as a potential downstream effector of Mac-SPP1hi. In vitro assays were conducted to examine the regulatory role of SPP1 on the Gal-1-FAK/MAPK signaling axis and its effects on macrophage migration. A co-culture system was used to assess the activation of fibroblasts by Mac-SPP1hi. In vivo, a rat model of tracheal injury was established, and SPP1 gene knockdown materials specifically targeting macrophages at the injury site were applied to evaluate alterations in local inflammation and airway stenosis, thereby elucidating the functional significance and molecular mechanism of the Mac-SPP1hi axis in BTS. Results scRNA-seq analysis revealed significant enrichment and abnormal activation of Mac-SPP1hi cells in BTS granulation tissue, with marked upregulation of pathways related to inflammation and cell migration. Multi-omics integration pinpointed Gal-1 as a critical downstream target. Functional experiments demonstrated that Mac-SPP1hi promoted macrophage migratory capacity and inflammatory cytokine secretion via Gal-1-mediated activation of the FAK/MAPK pathway. Knockdown of SPP1 or pharmacological inhibition of Gal-1 attenuated macrophage migration and reduced inflammatory responses. Mac-SPP1hi also enhanced fibroblast migration and increased deposition of α-SMA and Col1A1. In the rat model, targeted SPP1 knockdown in macrophages markedly reduced local inflammation, limited granulation tissue formation, and alleviated airway stenosis. Histological analysis confirmed decreased inflammatory infiltration and mitigated aberrant tissue repair. Conclusion This study is the first to systematically elucidate the molecular regulatory mechanisms of Mac-SPP1hi in BTS-associated inflammation and aberrant repair, revealing its activation of the SPP1-Gal-1-FAK/MAPK axis to drive inflammatory progression. These findings highlight Mac-SPP1hi as a potential therapeutic target, offering new strategies for the prevention and treatment of BTS. This abstract is funded by: None
Liu et al. (Fri,) studied this question.