Fibroblast heterogeneity links inflammatory microenvironments to tissue remodeling in chronic rhinosinusitis.

BACKGROUND: Chronic rhinosinusitis (CRS) is a heterogeneous inflammatory disorder with distinct endotypes and tissue remodeling. Fibroblasts contribute to extracellular matrix remodeling and immune regulation, but their heterogeneity in CRS remains unclear. METHODS: Public single-cell transcriptomic data from 21 CRS and healthy control samples were integrated with bulk RNA sequencing data from primary fibroblasts derived from nasal polyps stimulated with phosphate buffered saline (PBS), interferon (IFN)-γ, interleukin (IL)-4, or IL-17A. Fibroblast subsets, pseudotime trajectories, and inferred communication between fibroblasts and immune cells were analyzed. Cytokine-induced signatures were projected onto the fibroblast atlas. A subset of longitudinal RNA-seq data from the COMPASS cohort (NCT05598424) was used to validate predefined candidate marker genes, including IL-4-associated markers of the C8 subset and IL-17A-associated markers of the C5/C10 subsets, during glucocorticoid treatment across V1 (baseline), V2 (after 4 weeks of intranasal corticosteroid treatment), and V3 (after subsequent systemic glucocorticoid therapy). Temporal expression changes were assessed using linear mixed-effects models with V1-V2 and V2-V3 comparisons. RESULTS: Twelve transcriptionally distinct fibroblast subsets (C0-C11) were identified across CRS subtypes. C8 fibroblasts were enriched in eosinophilic CRSwNP and associated with type 2 inflammation related genes, including CCL26 and POSTN, whereas C5 and C10 were associated with non-eosinophilic CRSwNP and IL-17A related inflammatory features. Pseudotime analysis suggested two trajectories from C4 toward C8 and C10 respectively. CellChat revealed bidirectional communication between fibroblasts and immune cell subsets. Cytokine stimulation induced distinct transcriptional responses: IFN-γ mainly induced antigen presentation and interferon response; IL-4 induced contraction and cytoskeleton related features; and IL-17A activated IL-17, nuclear factor κB, and tumor necrosis factor signaling pathways. Signature projection linked IL-4 related features mainly to C8 fibroblasts and IL-17A related features mainly to C5 and C10 fibroblasts. Longitudinal analysis of glucocorticoid-treated patients identified a stable C8-associated transcriptional signature characterized by sustained downregulation of CCL26, POSTN, CISH, and CCL13 across V1-V3 time points. CONCLUSIONS: Fibroblasts in CRS exhibit marked heterogeneity and may contribute to tissue remodeling and immune regulation in response to distinct inflammatory stimuli. The IL-4-associated C8 state and IL-17A-associated C5/C10 states may represent distinct fibroblast states associated with different CRS inflammatory microenvironments. C8-associated transcriptional module shows stable and sustained suppression under glucocorticoid treatment.