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A2462 - GM-CSF Drives Alveolar Regeneration from Distal Lung Stem Cell Pools After Influenza Virus Infection
Author Block: A. I. Vazquez1, M. Heiner1, E. El Agha2, S. Bellusci2, T. Hain3, W. Seeger1, S. Herold1; 1Universities of Giessen and Marburg Lung Center (UGMLC), Justus Liebig University, part of the German Lung Center (DZL), Giessen, Germany, 2Excellence Cluster Cardio-Pulmonary System (ECCPS), Giessen, Germany, 3Justus-Liebig-Universitat Giessen, Giessen, Germany.
Rationale: Severe influenza virus (IAV) pneumonia is characterized by extensive epithelial cell death, edema, and inflammation causing acute lung injury. In order to regain homeostasis, restoration of the alveolar epithelial barrier is achieved by coordinated mechanisms involving the activation of stem/progenitor cell proliferation and differentiation. GM-CSF (granulocyte-macrophage colony stimulating factor) is a well-known growth factor which has been shown to promote proliferation of alveolar epithelial type 2 cells (AECII) after lung damage. Based on these observations, the aim of this study was to investigate the impact of GM-CSF on different distal lung stem/progenitor cell pools in tissue homeostasis and after influenza virus induced lung injury.
Methods: For in vitro experiments, our recently established bronchioalveolar lung organoid (BALO) model comprised of differentiated airways and alveoli, and fully representing the 3D organization of the distal lung, was employed. For this purpose, a well-characterized cell sorting strategy for the definition of bronchioalveolar stem cells (BASC) (EpCAMhighCD24lowitgα6highitgβ4highCD200+Sca1+SCGB1A1+SFTPC+) and resident mesenchymal cells (rMC) (CD31-CD45-EpCam-Sca-1+) was exploited. Accordingly, BASC and rMC were isolated from adult wild type (WT) C57BL/6, GM-CSF-overexpressing or -deficient/receptor deficient mouse lungs by high-speed cell sorting, and either cultured in Matrigel for evaluation of BASC self-renewal and cell differentiation capability or lysed for transcriptome profiling by RNA-Seq. For in vivo IAV experiments, WT, GM-CSF-overexpressing or -deficient mice were used. Mice were infected with influenza virus PR8 and renewal capacity of stem/progenitor cell populations, such as AECII and BASC, was analyzed by FACS and microscopy.
Results: Notably, the BALO model showed that the GM-CSF/GM-CSFR axis was necessary for organoid outgrowth, survival, and for proper alveolarization. In concordance, gene expression of important transcription factors involved in distal lung specification, sex determining region Y-box 9 (sox9) and retinoic acid receptor beta (Rarβ), were downregulated in GM-CSF-deficient BASC. Furthermore, 7 days post-infection with influenza, AECII numbers and BASC proliferation were considerably reduced in GM-CSF-deficient mice while the same parameters were found increased in mice overexpressing GM-CSF.
Conclusions: These data indicate that the presence of GM-CSF in the alveolar epithelium is indispensable for BASC proliferation and differentiation into AECII after viral infection, suggesting that GM-CSF may represent a distinct regenerative signaling pathway that can drive distal lung tissue repair upon lung injury.