Defining the mechanisms of injury resistance and role in restoring mucociliary transport of the airway hillock

About This Grant

PROJECT SUMMARY / ABSTRACT The airway epithelial surface is constantly exposed to the environment and serves as a barrier to many types of injury - toxins, infections, allergens, and other insults. The airway epithelial surface must regenerate after denuding injury. Both these aspects – barrier function and regeneration – can become dysregulated in diseases such as asthma, COPD, and bronchiectasis. We have discovered a new airway epithelial cell type, called the hillock, which survives various airway injuries and regenerates the epithelial surface to restore mucociliary transport. The overall hypothesis of this proposal is that the hillock plays a critical role in barrier function and regeneration in the airway and may become dysregulated in disease. Through single cell RNA sequencing and histological characterization, we find that the hillock highly expresses keratin 13 and desmoglein 3. These proteins are important in barrier function in of the skin, and we hypothesize that they similarly play a major role in the airway hillock. In Specific Aim 1 of this proposal, we will define the mechanism of hillock injury resistance. Using a hillock specific driver mouse we have developed, we will test this hypothesis by disrupting keratin 13 and desmosomes and assess for injury resistance by live/dead staining, proliferation, and electron microscopy. Hillocks also play an essential role in airway regeneration. We find when stimulated with retinoic acid, hillocks regenerate ciliated cells. In Specific Aim 2 of this proposal, we define the mechanism of ciliated cell differentiation from hillocks and restoration of mucociliary transport. It has been shown that different retinoic acid receptors may affect ciliogensis and oncogenesis. We hypothesize that retinoic acid receptor alpha and best leads differentiation of hillocks into ciliated cells which could then restore mucociliary transport. We will test this hypothesis in both mouse and human hillock explants. After performing luminal injury, we will regenerate the epithelia in the presence of retinoic acid receptor selective agonists and characterize ciliogenesis by immunocytochemistry, electron microscopy, cilia length measurements and functional assessment by micro-optical coherence tomography. This work is potentially paradigm shifting as it implicates a specific cell type, hillock cells, in disease pathology of multiple airway diseases and interrogates this cell type to uncover novel therapeutic targets. Dr. Shah will carry out this work under the mentorship of Dr. Jay Rajagopal, a leader in stem cell biology with an outstanding record of guiding young investigators to independence. Through this proposal, Dr. Shah will obtain training in (1) inducible genetic models, (2) developmental and stem cell biology, (3) explant models of disease, and (4) functional real-time imaging, which is only possible in the environment of the Rajagopal lab and collaborators at MGH. An advisory team of complementary and diverse scientists have been assembled to provide breadth and depth to the training plan. Hands-on training will be supplemented with coursework in stem cell biology and advanced imaging, necessary for Dr. Shah to become an independent R01 funded investigator.