The goal of the proposal is to determine the mechanism by which the long non-coding RNA (lncRNA) WFDC21P controls response of the airway epithelium to IL13. In allergic asthma, type 2 cytokines, such as IL13, signal through STAT (Signal Transducer and Activator of Transcription) proteins to drive pathophysiologic changes in the airway epithelium, including increased mucus production, goblet cell metaplasia, and loss of ciliated cells. Yet how these signals are coordinated is poorly understood. We have identified a lncRNA that coordinates the response of the airway epithelium to IL13 and hypothesize that it provides a mechanistic link between IL13 signaling and asthmatic lung pathology. In an air liquid-interface organoid culture system of primary human bronchial epithelial cells (BECs)?that recapitulates much of the in vivo physiology of the airway epithelium? WFDC21P is the most significantly induced lncRNA following IL13 exposure. WFDC21P deficient BECs show increased STAT signaling following IL13 exposure, implicating this lncRNA in controlling the pathophysiology of pulmonary type 2 immune responses. Importantly, WFDC21P is overexpressed in nasal brushing of patients with asthma. We hypothesize that WFDC21P regulates the response of airway epithelial cells to IL13 by promoting STAT dephosphorylation. Understanding how WFDC21P controls responses of the airway epithelium to type 2 inflammation could potentially identify new tissue-specific targets for therapeutic intervention.
In Aim 1 we will use single-cell RNA-Seq analysis of organoid cultures to determine which cell types upregulate WFDC21P following IL13 exposure. Next, using ATAC-Seq, ChIP-Seq and knockdown approaches, we will identify which transcription factors control WFDC21P expression.
In Aim 2 we will determine which cell types require WFDC21P to drive IL13-mediated asthma pathology. Using both knockdown and overexpression of WFDC21P we will measure changes in the differentiation, cellular composition, mucociliary status, and chemokine production of BEC organoid cultures grown in the presence or absence of IL13. Cultures will be evaluated by imaging mass cytometry, ATAC-Seq and single-cell RNA-Seq.
In Aim 3 we will identify the mechanism used by WFDC21P to regulate epithelial STAT signaling. We will test which STATs are impacted by loss of WFDC21P, and by using ChIP-Seq we will elucidate whether loss of WFDC21P impacts binding of STAT6 and STAT3 to target genes. Using RNA-immunoprecipitation (RIP) and an unbiased mass spectrometry based approach we will determine whether WFDC21P binds directly to STAT proteins and/or identify additional proteins bound to this lncRNA. Impact: This work will reveal fundamental molecular mechanisms driving pathological changes seen in allergic asthma, and will provide unprecedented resolution into cell type specific transcriptional changes following IL13 exposure. The experiments outlined here will establish a new role for lncRNAs as central regulators of airway epithelial cell biology, and as potential therapeutic targets for asthma.

Public Health Relevance

Inflammation in asthma alters the function of cells lining the airways making it harder to breath and driving susceptibility to infections. We have identified a long noncoding RNA (lncRNA) that regulates the response of lung cells to this inflammation. lncRNAs do not make proteins but have other functions in the cell, including the control of cell signaling. In this proposal, we will determine what activates expression of this lncRNA, and how it then orchestrates the response of lung cells to inflammation; this will provide a link to how inflammation causes the symptoms of asthma and may uncover new pathways for therapeutic targeting.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI141609-03
Application #
10075120
Study Section
Lung Cellular, Molecular, and Immunobiology Study Section (LCMI)
Program Officer
Minnicozzi, Michael
Project Start
2019-01-01
Project End
2022-12-31
Budget Start
2021-01-01
Budget End
2021-12-31
Support Year
3
Fiscal Year
2021
Total Cost
Indirect Cost
Name
Jackson Laboratory
Department
Type
DUNS #
042140483
City
Bar Harbor
State
ME
Country
United States
Zip Code
04609