Multi-Modal Analysis of Cell Populations and Architectural States Mediating the Progression and Resolution of Pulmonary Fibrosis

Introduction: Pulmonary fibrosis is a significant clinical burden that is often idiopathic and progressive. Nevertheless, the temporal progression of pulmonary fibrosis remains largely unexplored, and no studies to date have analyzed reparative cell populations at single-cell resolution. Thus, we performed a multi-modal analysis combining machine learning models of fibrotic/reparative pulmonary architecture with single-cell RNA sequencing (scRNAseq) of associated cell populations. Methods: C57BL/6 mice were intranasally instilled with 1.25mg bleomycin/mg body weight. At each timepoint, lungs were stained (Picrosirius red) and analyzed using an unsupervised machine learning algorithm for ultrastructural analysis (>2,500 images, 294 local/global fiber features), which were processed by uniform manifold approximation and projection and fitted to a minimum spanning tree-based pseudotime model. Individual lungs were imaged by microcomputed tomography, quantified by standard collagen assay, and digested using collagenases/RBC lysis buffer for scRNAseq. Results: Pulmonary matrix progressed from baseline to aberrant architecture after bleomycin administration, with peak pseudotime (fibrotic progression) at POD 21-28 and return to baseline by POD 42. Similar trends were observed in collagen concentration and lung radiological density. Baseline, fibrotic, and reparative lungs (PODs 0, 14, 35) presented variable cell populations, with significant shifts in composition of fibroblast subpopulations. Smooth muscle cells and matrix/ECM-modulating fibroblasts dominated the actively fibrotic state, while chemotactic and immunomodulatory fibroblasts emerged during active repair. Conclusion: Distinct cell subpopulations mediate the progression of fibrotic pulmonary architecture, with a transition from classical matrix-modulating fibroblasts to immune-reactive fibroblasts during the evolution of active fibrosis to post-fibrotic repair.