CTSK+ macrophages drive fibrotic stenosis in benign airway stenosis via the PROS1-AXL pathway

Background: Benign airway stenosis (BAS) involves progressive pathological narrowing of the trachea and main bronchi, causing clinically significant respiratory impairment that can advance to life-threatening obstruction. While fibrosis arises from dysregulated immune-stromal crosstalk, the specific cellular and molecular drivers of BAS remain poorly understood.

Methods: We conducted single-cell RNA Sequencing on clinical specimens representing the BAS spectrum normal airway, granulation tissue, and fibroproliferative tissue. Integrated bioinformatic analyses delineated cellular heterogeneity, intercellular communication, and differentiation trajectories. Spatial validation of key subsets was performed using multiplex immunofluorescence. Functional assessment of the PROS1-AXL axis involved treating primary human airway granulation fibroblasts (PHAGF) isolated from BAS patients with either conditioned medium from RANKL-stimulated THP-1 macrophages or recombinant PROS1, followed by inhibition with the AXL-specific antagonist R428. In parallel, an in vivo mouse model of BAS was used to evaluate the therapeutic efficacy of R428.

Results: Transcriptomic analysis revealed substantial remodeling of the BAS microenvironment, marked by epithelial depletion and expansion of stromal and immune compartments. We identified a novel macrophage subset co-expressing CTSK and SLC9B2, specifically enriched in granulation tissue. Communication analysis demonstrated selective PROS1-AXL signaling between CTSK⁺ macrophages and CD82⁺ fibroblasts. Pseudotemporal analysis positioned this crosstalk upstream of myofibroblast differentiation. Multiplex immunofluorescence confirmed CTSK⁺ macrophage localization in human BAS granulation tissue. In vitro, conditioned medium from RANKL-primed THP-1 macrophages promoted fibroblast activation in PHAGF cells through the PROS1-AXL axis, an effect that was attenuated by Axl inhibition with R428. Furthermore, Western blot analysis revealed that PROS1-AXL signaling activated the Akt/GSK3β pathway in PHAGF cells. In vivo, systemic administration of R428 in a BAS mouse model significantly reduced fibrotic remodeling, as evidenced by decreased granulation tissue hyperplasia, Collagen deposition, and improved survival compared to vehicle-treated controls. Furthermore, TNFSF11 on CD82⁺ fibroblasts may bind TNFRSF11A on CTSK⁺ macrophage precursors to drive their differentiation.

Conclusion: This work defines a pro-fibrotic cellular module in BAS CTSK⁺ macrophages and CD82⁺ fibroblasts interacting via PROS1-AXL and establishes a rationale for targeting this pathway, supported by both patient-relevant primary cell and in vivo evidence, to disrupt fibrosis and mitigate recurrence.