Alveolar epithelial cell plasticity and injury memory in human pulmonary fibrosis

Key results

1. ShownSingle-nucleus RNA-seq of human IPF and control lung cores identifies Alveolar Intermediate (ATi) cells that bridge alveolar type I (ATI), alveolar type II (ATII), and Aberrant Basaloid cell states and were under-recovered in prior cell-dissociation scRNA-seq studies.

   Across 54 control and IPF cores, ATi cells accounted for a median 3.87% of IPF epithelial nuclei versus 1.32% (p = 0.0015) and 2.17% (p = 0.038) in two prior scRNA-seq datasets, co-occurring strongly with Aberrant Basaloid cells (Spearman's rho = 0.813) and connecting ATI/ATII to Aberrant Basaloid cells in PAGA graphs (edge confidence > 0.2). · A “Missing Link” Between Aberrant Basaloid and…; Composition of Abnormal Epithelial Cells in IPF; The Continuum of States Between ATI, ATII and…; Figure legends

2. ShownNeither Aberrant Basaloid nor ATi cell proportions correlate with microCT tissue surface density in IPF cores, whereas ATII cells decline with worsening fibrosis.

   Across differentially fibrotic IPF lung cores, declining microCT surface density was significantly associated with reduced ATII cell proportions (p = 5.34e-4) and increased secretory (p = 0.013) and ciliated (p = 0.023) cells, while Aberrant Basaloid (median 2.72% to 2.91% across 3 datasets) and ATi proportions showed no significant association with surface density. · Composition of Abnormal Epithelial Cells in IPF; Figure legends

3. ShownIn disease-free human precision-cut lung slices (PCLS), rare proliferating Aberrant Basaloid-like cells emerge during normal ex vivo repair, whereas fibrotic cocktail (FC) stimulation drives massive expansion of cell-cycle-arrested, disease-like Aberrant Basaloid cells.

   From 72 hours onward in PCLS, FC exposure increased Aberrant Basaloid abundance from an average of 1.25% of epithelial cells in controls to 30.4% (t-test p = 0.004385) while reducing overall epithelial mitosis from 6.3% to 3.8% (p = 0.010607) and mitotic Aberrant Basaloid cells from 32.6% in controls to 0.49% in FC (p = 0.04077). · Longitudinal Assessment of ex vivo Fibrosis; Comparing control, fibrotic cocktail and IPF…; Figure legends

4. ShownSingle-nucleus MultiOme profiling reveals that IPF Aberrant Basaloid cells possess a distinct chromatin accessibility landscape dominated by occupied AP-1 transcription factor binding motifs, indicative of epigenetic injury memory.

   Across 3 control and 7 IPF lungs, the top 15 enriched transcription factor motifs in Aberrant Basaloid cells (p < 1E-20) shared the AP-1 consensus sequence (TGA G/C TCA), which appeared in >50% of Aberrant Basaloid differentially accessible peaks versus 12.5% of all merged peaks (hypergeometric p < 1e-50; median cell AP-1 z-score 5.29 vs 1.09 in ATi; Wilcoxon p = 2.2e-16) with active footprint dips. · Chromatin Accessibility Patterns of Aberrant…; AP-1 Transcription Factor Accessibility is a…; Figure legends