Alveolar macrophages drive tissue organization and regeneration in engineered whole lungs ex vivo

Introduction: : Lung diseases, including COVID-19 infection, chronic obstructive pulmonary disease (COPD), lung cancer, and tuberculosis, are leading causes of death worldwide, accounting for over 16 million deaths combined in 2021 (World Health Organization, 2024). Such significant global health burden calls for innovative modalities for studying lung biology and uncovering potential routes for new regenerative therapies. The role of immune cells in lung tissue organization and regeneration, beyond their canonical response to pathogens, is not well understood and challenging to study in vivo. We present an engineered whole-lung tissue model that newly incorporates epithelium, endothelium, mesenchyme, and immune cells, to study the impact of alveolar macrophages on lung regeneration ex vivo.

Materials and

Methods: : The engineered model utilized decellularized rat lungs as a whole-organ scaffold (Fig. 1A), which was recellularized with primary rat lung cells (Fig. 1B-C) and cultured in a bioreactor under controlled conditions, including vascular perfusion of oxygenated media and negative pressure cyclic stretch to mimic breathing (Fig. 1D). Fluid pressures were measured continually throughout culture at all inlets and outlets of the organ for subsequent mechanical analyses. Here we compared tri-culture (epithelium + endothelium + mesenchyme, n=6) to quad-culture lungs (epithelium + endothelium + mesenchyme + macrophages, n=6) to study the effects of adding macrophages to the system. Engineered lungs were analyzed by direct mechanical measurements, histology, and single-cell RNA sequencing (scRNAseq).

Results, Conclusions, and Discussions:: Here we present multicellular engineered lungs that are unprecedented in cell coverage, viability, regenerative phenotype, mechanical function, and methodologic replicability, compared to previous versions of this technology. We found that tri-culture lungs displayed fibroblast overgrowth (by histology/IF, PDGFRα-high), which was significantly reduced in quad-culture lungs, possibly through Fas signaling from alveolar macrophages (Fig. 1E-H). Tissue architecture and cell phenotype became more native-like with the addition of alveolar macrophages, including the emergence of bronchoalveolar stem cells (Sftpc+/Scgb1a1+), increased expression of lipofibroblast markers (Plin2, Thy1), and increased expression of capillary endothelial markers (Aplnr, Kdr, Vegfa) (Fig. 1I). Cell-cell signaling (ligand-receptor expression) within engineered lungs showed activation of several developmental and regenerative pathways with the addition of alveolar macrophages, including canonical WNT, Notch, and FGF (Fig. 1J). Based on fluid pressure measurements and microvascular modeling, the presence of supporting mesenchyme enhanced vascular recruitment (Fig. 1K), and adding alveolar macrophages significantly improved alveolar barrier function (Fig. 1L).
The addition of alveolar macrophages to multicellular engineered whole-lungs significantly improved tissue organization, cellular differentiation, signaling, and mechanical function, thereby suggesting a critical role for immune cells in lung regeneration (Fig. 1M). The impact of this work is the demonstration of a non-canonical role for immune cells in tissue organization and characterization of their role in lung regeneration, which could offer new routes for therapeutically reversing pathologic tissue remodeling in myriad diseases.

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