Abstract Rationale Childhood interstitial and diffuse lung disease (chILD) is a diverse group of rare pulmonary disorders. Although the genetic causes have been identified for several chILD disorders, the molecular underpinnings of most remain unclear, leading to diagnostic and therapeutic uncertainty. To address this, we are combining snRNA-seq and spatial transcriptomic approaches to build a comprehensive chILD cell atlas. Here we share the results of an initial analyses of snRNA-seq from 30 chILD cases. Methods Frozen residuals of clinically obtained lung biopsies, surgical resections, or explants were identified for inclusion from patients with inflammatory forms of chILD, prioritizing those with an established genetic etiology. Nuclear extraction and processing were done on the 10x GenomicsTM platform per protocol. Quality control included metrics of cellular mitochondrial percentage and RNA count. A UMAP was generated using Human-LungMAP core cell type definitions. Unsupervised hierarchical clustering and gene set enrichment analysis (GSEA) were conducted for Type II pneumocyte (AT2) and macrophage cell populations separately to screen for evidence of convergent downstream expression signatures among subsets of chILD of varying underlying cause. Detailed methods are reported in a separate analysis pipeline abstract. Results We analyzed 341,000 nuclei from 33 chILD patients and identified all expected lung cell lineages. Unsupervised hierarchical clustering of macrophage profiles revealed that macrophages from SFTPB and ABCA3-related chILD grouped with lysosomal disorders (NPC2, GRN) and JAK-STAT pathway variant cases. AT2 cells from ABCA3 cases clustered most closely with GRN, NPC2 and STAT1-associated chILD. Gene set enrichment analysis demonstrated increased interferon-α/γ signaling in both macrophages and AT2 cells from ABCA3 cases compared to SFTPB, with parallel upregulation of JAK-STAT signaling in macrophages. Discussion To our knowledge, this represents the first single-nucleus transcriptomic dataset generated in chILD. Early analysis suggests that surfactant-related disorders exhibit distinct immune-epithelial signaling states, including interferon-α/γ and JAK-STAT activation in ABCA3 deficiency. Spatial validation and integration of 15 additional chILD cases are underway to define cell-cell signaling networks and therapeutic targets in pediatric diffuse lung disease. This abstract is funded by: The Chan-Zuckerberg Initiative
Kennedy et al. (Fri,) studied this question.