Integrated Multi-Omics Identifies SLIT2 as a Regulator of Senescence-Associated Fibroblast Remodeling in Idiopathic Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease that is strongly associated with aging. How aging contributes to fibroblast state remodeling and intercellular signaling dysregulation in IPF remains poorly defined. We integrated bulk transcriptomics, single-cell RNA sequencing, spatial transcriptomics, and DNA methylation data from human IPF lungs to characterize fibroblast heterogeneity, trajectory dynamics, intercellular communication, and epigenetic aging. SLIT2 downregulation and senescence-associated changes were then examined in a bleomycin (BLM)-induced mouse model of pulmonary fibrosis. The effects of SLIT2 on fibroblast senescence and paracrine pro-fibrotic activity were assessed in BLM-treated MRC-5 fibroblasts using recombinant SLIT2 supplementation and siRNA-mediated knockdown. In an independent DNA methylation cohort, epigenetic aging clocks and mediation analyses in both directions were used to examine statistical relationships among epigenetic age acceleration, SLIT2 expression, and IPF risk. These analyses identified five fibroblast subtypes, with TCF21⁺ fibroblasts predominating in normal lung and CTHRC1⁺ fibroblasts markedly enriched in IPF. Trajectory analyses inferred a potential fibroblast state trajectory from homeostatic TCF21⁺ toward pathological CTHRC1⁺ fibroblast states, along which a senescent TCF21⁺ subpopulation showed preferential trajectory bias toward the CTHRC1⁺ endpoint. SLIT2 was preferentially expressed in TCF21⁺ fibroblasts, which represented an important predicted sender population within the SLIT-ROBO communication network. In a BLM-induced mouse model, fibrotic remodeling was accompanied by increased senescent cell burden and SLIT2 downregulation, consistent with the observations in human IPF. In vitro, rhSLIT2 attenuated BLM-induced senescence and pro-fibrotic paracrine activation, whereas SLIT2 knockdown exacerbated both. SLIT2 manipulation also altered NF-κB signaling and SASP-associated responses, supporting a regulatory role for SLIT2 in senescence-associated inflammatory and pro-fibrotic remodeling. In an independent DNA methylation cohort, SLIT2 promoter hypermethylation co-occurred with multi-dimensional epigenetic aging acceleration and was inversely correlated with SLIT2 expression. Mediation analyses suggested potential reciprocal associations between epigenetic aging acceleration and SLIT2 silencing in relation to IPF risk. Overall, this study identifies an aging-associated fibroblast remodeling pattern in IPF, characterized by senescence-associated TCF21⁺ fibroblasts and an inferred relationship toward pathological CTHRC1⁺ fibroblast states. SLIT2 emerged as a regulator of TCF21⁺ fibroblast homeostasis, with its reduction associated with NF-κB/SASP activation and pro-fibrotic phenotypes. Epigenetic aging acceleration and SLIT2 dysregulation were associated with IPF risk, highlighting SLIT2 as a potential therapeutic target in aging-associated pulmonary fibrosis.