In the previous DFG project we have described and characterised chemosensory brush cells in various organs. For tracheal brush cells we could show, that they are critical for sensing the micro-environment in the airways. A stimulation of brush cells with bacterial or bitter-tasting substances led to a direct ACh release and local protective immunological responses such as an increase in mucociliary clearance and induction of neurogenic inflammation. In the follow-up project we would like to focus on:I. The signal transmission between brush cells and sensory nerve fibers: synapses vs. volume transmission. Do brush cells receive an input from the CNS?II. The origin of brush cells and their turn over rate? III. Characterization from brush cells in the human respiratory system.For the characterization of the communication between brush cells and sensory nerve endings, we will use a modified pseudo-typed rabies virus (for retrograde infection) and a modified vesicular stomatitis virus (for anterograde infection). The functional proof for the activation of brush cells and the transmission to the nerve fibers will be conducted in vivo on TRPM5-GCaMP and Pirt-GCaMP3 mice using 2P-microscopy Ca2+-Imaging. For control experiments we will use Pirt-GCaMP3/TRPM5-DTA mice, which lack brush cells and TRPM5-DREADD in which brush cells are activated. To determine their origin of brush cells and to study their development we will specifically deplete tracheal brush cells using a TRPM5-DTR-tauGFP mice and inhalative application of dephtheria toxin. The number of cells will be quantified at day 1, 3, 7, 14 and 21 after successful depletion. In collaboration with Dr. A.-E. Saliba (Helmholtz Institute for RNA-based infection research, HIRI, Wuerzburg) we will perform a combined analysis of all time-points by single-cell RNA-seq and computational analysis to cluster the cells. In order to trace the development of brush cells along the time-course, data will be integrated using Seurat and the developmental path will be inferred using time-resolved computational approaches. We will perform a time course experiment in duplicates representing a total of 10 different time-points sampled and aiming for 1500 cells per time points. Next, we aim to prove the functionality of the newly-developed brush cells. ACh release and activation with known agonists will be used as a read-out for the functional activity. Lastly, we will systematically study the localization and characterization of brush cells and the classical components of the canonical taste transduction cascade in the human airways. We expect to gather new insights into brush cell physiology (sensing, transmission and regeneration), which may offer a new therapeutic opportunity for the treatment of pneumonia and respiratory diseases.
|Effective start/end date||01.01.12 → 31.12.18|
In 2015, UN member states agreed to 17 global Sustainable Development Goals (SDGs) to end poverty, protect the planet and ensure prosperity for all. This project contributes towards the following SDG(s):