B76-21 Comparative Single-cell Trajectory Analysis of COPD and Healthy Lungs Using Monocle3

Background

Monocyte-to-macrophage differentiation is strongly shaped by local tissue cues; however, how this process is altered in chronic disease and the extent to which diseased microenvironments actively reprogram myeloid fate remain poorly defined. This is particularly relevant in chronic obstructive pulmonary disease (COPD), where persistent monocyte-driven inflammation and macrophage dysfunction are central to disease pathogenesis. We hypothesized that single-cell trajectory analysis could resolve monocyte-to-macrophage differentiation pathways in the human lung and reveal how COPD alters gene expression dynamics along these trajectories.

Methods

We reanalyzed single-cell RNA sequencing data from subjects with COPD and healthy control donor lungs across multiple independent datasets. Cells were processed using Seurat and ordered along differentiation trajectories using Monocle3, with low-dimensional embeddings generated by PHATE. Monocyte-to-IM and IM-to-AM transitions were isolated and aligned along pseudotime. Genes dynamically regulated along these trajectories and modified by disease status were identified using two complementary approaches. Moran’s I-based trajectory analysis was used to detect genes with spatially autocorrelated expression along pseudotime, and generalized linear models were applied to quantify the independent and interactive effects of pseudotime and disease status on gene expression. Genes meeting significance thresholds (FDR < 0.05) were subjected to functional annotation using Gene Ontology and KEGG pathway enrichment analyses via Enrichr.

Results

Among the analyzed subjects, 11 subjects had emphysema and 50 did not. Trajectory inference revealed divergent monocyte-to-macrophage differentiation programs in healthy versus COPD lungs. In healthy samples, 341 genes were significantly upregulated along pseudotime, whereas 1,203 genes were preferentially upregulated along COPD trajectories. Genes enriched in healthy trajectories included RAB31, PCNX1, NEK7, ACSL3, NAMPT, NFAT5, SLC44A1, and TLR2. Functional enrichment analysis indicated overrepresentation of lipid and arachidonic acid binding, complement activation, and cytokine receptor activity, consistent with coordinated metabolic regulation and immune signaling during homeostatic macrophage differentiation. In contrast, COPD-associated trajectories were marked by increased expression of inflammatory, stress-response, and antigen-presentation genes, including NLRP3, SAMSN1, PLCB1, ACSL1, IL1B, EREG, MT-CO3, FTL, and CD74.

Conclusions

These findings demonstrate that the COPD lung microenvironment fundamentally alters the trajectory of monocyte-to-macrophage differentiation, shifting gene expression dynamics away from homeostatic metabolic and signaling programs toward persistent inflammatory and stress-associated states. This disease-specific rewiring of macrophage differentiation provides a mechanistic framework linking chronic lung injury to sustained myeloid dysfunction in COPD.

This abstract is funded by: None