Caffeine Citrate Ameliorates Hyperoxia-Induced Alveolar Arrest by Targeting Macrophage-Driven TGF-β1 Signaling and Restoring DATP Differentiation

Abstract

Bronchopulmonary dysplasia (BPD) is primarily characterized by arrested alveolar development and structural simplification. Normal alveolarization relies on the orchestrated differentiation of alveolar type II (AT2) into type I (AT1) epithelial cells. However, during BPD pathogenesis, aberrant macrophage-epithelial crosstalk disrupts this trajectory, leading to a profound accumulation of aberrant transitional cells. Although caffeine citrate, a first-line clinical therapeutic, is known to mitigate BPD, the core molecular mechanisms by which it alleviates this differentiation blockade and remodels the local microenvironment remain elusive. This study aims to delineate the stalled transitional state of alveolar epithelial cells and the aberrant macrophage-epithelial communication networks under hyperoxic stress, further elucidating the molecular mechanisms underlying the lung-protective effects of caffeine citrate. We integrated single-cell RNA sequencing (scRNA-seq), pseudotime trajectory analysis, and multiplex immunofluorescence to dissect the pathophysiology of BPD. Additionally, molecular docking and molecular dynamics simulations were employed to predict the potential therapeutic targets of caffeine citrate. Hyperoxia was associated with marked remodeling of the alveolar epithelial compartment, including relative depletion of AT2 cells and enrichment of DATP-like transitional epithelial states. Pseudotime analysis, KRT8 immunostaining, and increased expression of DATP-associated proteins, including KRT19, KRT8, ITGB6, and CDKN1A, supported persistence of a transitional epithelial program during hyperoxic injury. CellChat analysis predicted enhanced macrophage/monocyte-associated TGF-β signaling toward epithelial populations, while in vivo and in vitro experiments demonstrated increased TGF-β1 expression and Smad3 phosphorylation under hyperoxic conditions. Macrophage–epithelial co-culture further enhanced Tgfb1-associated responses. Caffeine citrate attenuated hyperoxia-associated Tgfb1/TGF-β1 and Smad3 signaling changes and partially restored epithelial-associated gene expression. In silico analyses suggested potential structural interactions between caffeine and TGF-β receptor-related targets. This study demonstrates that hyperoxia-induced aberrant macrophage-epithelial crosstalk constitutes the core mechanism driving the stagnation of alveolar epithelial cells at the DATP stage, consequently triggering alveolarization arrest. By disrupting this pathological communication network, caffeine citrate effectively reverses alveolar developmental arrest. These findings underscore that targeted intervention in the “macrophage-epithelium-DATP” microenvironmental axis represents a crucial translational strategy for the prevention and treatment of BPD in premature infants.

Keywords: Bronchopulmonary dysplasia; Hyperoxia; Caffeine citrate; Alveolar epithelial differentiation; Alveolar macrophages; TGF-β1/Smad3 signaling