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RATIONALE

Pulmonary arterial hypertension (PAH) is characterized by progressive pulmonary vascular remodeling and immune dysregulation; however, prior studies have emphasized expansion of inflammatory immune populations rather than loss of homeostatic immune programs. Interstitial macrophages (IMs) are myeloid cells within the lung interstitium implicated in immune regulation and vascular homeostasis, yet their abundance, functional state, and clinical relevance in advanced human PAH remain incompletely defined.

METHODS

Explanted lung tissue from patients with PAH undergoing lung transplantation and non-diseased donor controls from the Pulmonary Hypertension Breakthrough Initiative (PHBI) was analyzed using single-nucleus RNA sequencing (snRNA-seq; n=67) to define immune cell populations and transcriptional programs. Spatial transcriptomics (Xenium; n=73) was used to assess the spatial distribution of interstitial macrophages within lung tissue regions enriched for pulmonary vasculature. Bulk lung RNA sequencing data (n=148) were analyzed using cell-type deconvolution. These single-nucleus, spatial, and bulk transcriptomic datasets were derived from PHBI but comprised non-identical patient samples. Associations between IM abundance and hemodynamic measures obtained by right heart catheterization were evaluated using correlation analyses. Differential gene expression and pathway enrichment analyses were performed to define PAH-associated transcriptional programs within IMs.

RESULTS

Across modalities, PAH lungs demonstrated a consistent reduction in IM abundance compared with controls, including snRNA-seq, spatial transcriptomics, and bulk transcriptomic deconvolution. Interstitial macrophages from PAH lungs profiled by snRNA-seq exhibited coordinated downregulation of oxidative phosphorylation, mitochondrial respiration, and reparative gene programs, consistent with loss of a metabolically competent, homeostatic state. IM abundance by snRNA-seq was positively associated with cardiac output, such that lower IM levels corresponded to worse hemodynamics. Furthermore, lower IM levels inferred by bulk RNA-seq correlated with higher mean pulmonary arterial pressure and showed a similar trend with worse pulmonary vascular resistance index.

CONCLUSIONS

Multimodal single-cell, spatial, and bulk transcriptomic analyses demonstrate that advanced human PAH is characterized by depletion and functional collapse of a homeostatic interstitial macrophage compartment. Loss of a metabolically competent IM compartment is associated with worse pulmonary hemodynamics across modalities, highlighting failure of a homeostatic interstitial macrophage compartment—rather than immune expansion—as a defining feature of advanced PAH. These findings underscore the value of human lung multimodal profiling for linking cellular pathology to clinical severity in pulmonary vascular disease.