Mucociliary innate defense mechanism in the human distal airway - Project summary: This application responds to the Notice of Special Interest (NOSI) identified as NOT-RM-24-013, which supports replication studies using independent contract resources. The overarching goal of the original study, “Mucociliary Innate Defense Mechanism in the Human Distal Airway”, is to uncover mechanistic insights into region-specific mucociliary innate defense mechanisms, with a focus on the human small airways. Airway mucociliary clearance (MCC) is a critical innate defense system essential for maintaining lung health. Impaired mucus transport associated with mucus hyperconcentration is central to the pathogenesis of muco-obstructive lung diseases (MOLDs). However, the mechanisms that integrate regional ciliary and mucus properties into MCT remain poorly understood. This question is particularly important in small airways, which are the earliest and most severely affected regions in MOLDs. Our original study is designed to address the following questions: 1) How do small airways regulate MCT through mechanisms distinct from those of larger airways? 2) Why are small airways particularly vulnerable to mucus obstruction? 3) what mechanisms drive mucociliary failure in small airways in MOLDs? To address these questions, we developed microdissection techniques to establish human small airway epithelial (SAE) cell culture models, allowing for detailed characterization of region-specific MCC mechanisms. Data from our original study, utilizing single-nucleus (sn) RNA/ATAC-seq, identified a unique subset of secretory cells, termed distal airway specific secretory cells (DASCs), enriched in SAE cultures. DASCs are transcriptionally identical to recently characterized secretory cell population in human small airway tissues in vivo. While DASCs are known for their multipotent progenitor properties, their role in MCC remains understudied. Of clinical relevance, DASC loss has been documented as a common pathological feature of MOLDs, suggesting their critical role in maintaining small airway MCC function. Our snRNA/ATAC-seq data identified DASC-enriched chromatin regulatory elements, including transcription factor (TF) motifs associated with NKX2-1. Utilizing CRISPR/Cas9 technology in primary human SAE cultures, we demonstrated that NKX2-1 is required for maintaining DASC identity. Notably, disruption of NKX2-1-mediated transcriptional pathways resulted in pathological goblet cell metaplasia, mucus hyperconcentration, and MCT dysfunction. While our original snRNA/ATAC-seq data from three control donors identified key region- and cell-type specific TF motifs, an independent replication cohort with data from additional donors is essential to enhance scientific rigor, increase statistical power, and identify a more comprehensive set of TFs regulating region- and cell-type identity. We propose two specific aims for the replication study, including: 1) Validate region- and cell-type-specific chromatin regulatory elements in an independent replication cohort; and 2) Identify robust TF motifs regulating DASC identity and function. Successful completion of these aims will advance our understanding of MCC mechanisms in the normal lung and inform novel therapeutic strategies for MOLDs.
