# Spatial transcriptomics reveals altered communities and drivers of aberrant epithelia and pro-fibrotic fibroblasts in interstitial lung diseases

## TL;DR

The study uses advanced sequencing and spatial analysis to uncover cell changes in lung diseases, identifying key drivers of fibrosis and abnormal cell behavior.

## Contribution

The work identifies novel cell communities and molecular drivers, including CTHRC1-hi fibroblasts and NFATC4, in lung fibrosis using multimodal data integration.

## Key findings

- Fibrotic niches contain pro-fibrotic CTHRC1-hi fibroblasts and aberrant epithelial cells.
- TGF-β, IL-1β, and TNF-α together drive maladaptive epithelial cell differentiation.
- NFATC4 is required for pro-fibrotic gene induction in fibroblasts.

## Abstract

Interstitial lung diseases (ILD) are characterized by fibrotic scarring of the lung parenchyma with remarkably unfavorable prognosis. Using single-nucleus RNA sequencing and spatial transcriptomics, we generated a comprehensive cellular network of the distal lung and its alterations in fibrosis. Integration with histopathology revealed that the transformation of normal parenchyma into fibrotic tissue is accompanied by ectopic bronchiolization and decellularization. Areas of active fibrosis were characterized by co-localization of pro-fibrotic CTHRC1-hi fibroblasts and aberrant transitional epithelial cells. We modeled this maladaptive differentiation of alveolar epithelial cells using organoids, demonstrating that all three pro-inflammatory ligands present in this pathogenic niche, TGF-β, IL-1β, and TNF-α, are jointly required for their induction. Additionally, we identified a requirement for the transcription factor NFATC4 during myofibroblast differentiation driven by soluble factors or mechanosensing. Collectively, this work identifies essential molecular drivers of the cellular interactions underlying lung fibrosis.

•Multimodal atlas (snRNAseq/spatial/histology) reveals gradients in fibrotic activity•Fibrotic niches harbor CTHRC1-hi fibroblasts and aberrant epithelial cells•TGF-β, IL-1β, and TNF-α drive maladaptive aberrant transitional cell differentiation•Transcription factor NFATC4 regulates pro-fibrotic gene induction in fibroblasts

Multimodal atlas (snRNAseq/spatial/histology) reveals gradients in fibrotic activity

Fibrotic niches harbor CTHRC1-hi fibroblasts and aberrant epithelial cells

TGF-β, IL-1β, and TNF-α drive maladaptive aberrant transitional cell differentiation

Transcription factor NFATC4 regulates pro-fibrotic gene induction in fibroblasts

Using single-nucleus RNA sequencing and spatial transcriptomics, Jaiswal et al. identify disease-associated cell communities that include aberrant transitional alveolar epithelium and pro-fibrotic fibroblasts. Using in vitro culture systems, they validate molecular drivers of these pathogenic cell states, including pro-inflammatory cytokines and the transcription factor NFATC4.