Targeting p16INK4a in alveolar type 2 cells to induce endogenous lung alveolar regeneration

Introduction: Alveolar type 2 cells (AT2) play a key role in alveolar regeneration after alveolar damage. Mechanisms involved in this process are not well known. Aims and Objectives: Targeting p16INK4a, a cell cycle inhibitor, in AT2 could be a way to promote alveolar regeneration. Methods: In vitro alveolar organoids were obtained by co-culturing EpCAM+ cells (sorted with magnetic beads from lungs of p16INK4a-/- or wild type (WT) mice) with WT lung primary fibroblasts. Colony forming units (CFU), organoid size, organoid types (alveolar, bronchoalveolar by pro-SpC and ACT staining) were quantified at D14 and D21. Organoids from emphysematous mice (elastase model) were compared to organoids from PBS-injected mice as a control with the same genetic background (p16INK4a-/- or WT). In vivo, morphological analysis (mean linear intercept) and AT2 number were quantified in elastase or PBS injected mice at D21 and D90 to assess alveolar destruction and regeneration according to WT or p16INK4a-/- genotype. Results: Organoids made with p16INK4a-/- EpCAM+ cells were as numerous but had an increased size compared to those made from WT EpCAM+ cells at D14 and D21. CFU and organoid size from emphysematous mice were significantly decreased compared to control mice. Number and size of organoids made with p16INK4a-/- EpCAM+cells from emphysematous mice were higher than organoids made with WT EpCAM + cells from emphysematous mice. In vivo, p16INK4a deletion was associated with an increase of AT2 at D21 without a protective effect on alveolar architecture. At D90, p16INK4a-/- mice had less emphysema than WT mice. Conclusions: p16INK4a deletion increases endogenous alveolar regeneration by AT2 cells. Mechanisms involved remain to be determined.