DESCRIPTION (provided by applicant): Changes in the airway epithelium are prominent contributors to a number of human diseases, notably asthma. Our group has shown that the Th2 cytokine interleukin-13 (IL-13) drives characteristic alterations in epithelial gene expression leading to mucous metaplasia in mouse models and in a large subset of human subjects with asthma. I hypothesize that microRNAs (miRNAs) play an important role in these changes. In support of this, we have shown that there are significant differences in expression of miRNAs between healthy and asthmatic subjects. Since joining the Erle lab, I found evidence that many of these changes are driven by IL-13 and developed tools required to monitor, inhibit, or mimic effects of miRNAs in bronchial epithelial cells. In this revised proposal, I focus on testing the involvement of specific epithelial miRNAs in mucous metaplasia. The first aim is to determine if the repression of the miR-449/34 family is necessary and sufficient for IL-13- driven mucous metaplasia. Among the most significantly suppressed miRNAs are members of the miR- 449/34 family, which have known roles in multiciliogenesis through repression of Notch signaling. As Notch signaling also plays a key role in selection of secretory cell fate, I hypothesize that IL-13 repression of miR- 449/34 acts to increase Notch signaling and promote mucous metaplasia. I will test whether miR-449 is necessary and sufficient for IL-13 driven mucous metaplasia using normal human bronchial epithelial cells differentiating in an air-liquid interface culture. I will inhibit miR-449 using miRNA inhibitors and assess for markers of mucous metaplasia. I will use a miR-449 mimic in cells exposed to IL-13 and test for suppression of mucous metaplasia. I will characterize additional targets of this miRNA family that may act in concert with Notch signaling to promote mucous metaplasia. The second aim is to determine how selected miRNAs stimulated by IL-13 and upregulated in asthma are involved in mucous metaplasia and other epithelial abnormalities. I hypothesize that miRNAs upregulated in response to IL-13 have a role in mucous cell differentiation and function. I have identified several miRNAs for further characterization: miR-663a/1908, miR- 1275, miR-765, and miR-1228-5p. These miRNAs have significant upregulation in asthmatic patients versus healthy subjects and are also responsive to IL-13 in our cell culture system. I will first use in situ hybridization to localize the expressionof these miRNAs and use a lentiviral sensor system to determine their activity in mucous cells and other epithelial cells. I will use mimics and inhibitors to define their role in mucous metaplasia.In addition to lab work, I will attend relevant courses and seminars to continue to expand my knowledge base. I will be closely mentored by Dr Erle and a committee comprised of experts in the molecular and cellular biology of lung disease. This proposal will provide me with the tools and experience for future independent work as a research-oriented pulmonary physician-scientist.

Asthma is a major contributor to morbidity and mortality in the United States. In asthma, the epithelial layer of the airway undergoes dramatic changes. This proposal aims to elucidate the genetic basis of these changes, which will give us new tools to better diagnose and treat asthma and other diseases of the lung.