Project Summary:Allergic asthma is a chronic inflammatory disease that directly affects large American population with anestimated total annual healthcare cost of 20 billion dollars. The heterogeneous nature driven by differentunderlying disease processes makes it challenging to treat and control the asthma symptoms. Therefore, inaddition to having the effective disease prevention and management strategies, the development of novel andphenotype-specific therapeutic approaches is of great interest. Interleukin (IL)-13 has been recognized as acentral mediator of TH2-dominant asthmatic phenotypes including excess mucus production and airwayhyperresponsiveness. Consequently, therapeutics targeting IL-13 are very attractive with studies already inphase II clinical trials. But initial studies reported some adverse events associated with the use of IL-13targeting therapeutics because IL-13 also elicits other immunoregulatory effects. For example, IL-13suppresses TH17 cytokine production in an IL-10-dependent manner and thereby may play an important role inTH17-associated autoimmune diseases. Accordingly, patients receiving IL-13–targeted therapeutics hadsignificantly increased adverse events involving the musculoskeletal and autoimmune-related diseases.Therefore, identifying targets that specifically affect the proliferative and metaplastic activity of IL-13 is crucial.The current study proposes a novel approach that switches the proliferative IL-13 to one that causes cell deathin airway epithelial cells. IL-13 exerts proliferative effect by modulating various programmed cell deathpathways. The anti-apoptotic protein, Bcl-2 inhibits apoptosis directly by interacting with various pro-apoptoticBH3-domain effector proteins. Indeed, our preliminary studies showed that IL-13 induced the proliferation andsustained the hyperplastic mucous cells by upregulating Bcl-2 expression. Blocking Bcl-2 by shRNA-basedapproach and by a small molecule Bcl-2 antagonist, ABT-263 reduced the mucus expression and causedmucous cell death. Screening for the pro-apoptotic factors showed that IL-13 induced a BH3-domain onlyprotein, Bik, an epithelial cell death regulator in a STAT1-dependent manner. Thus, IL-13 modulates both Bcl-2and Bik, the anti- and pro- cell death proteins, respectively to alter the cell fate, and blocking Bcl-2 results in aBik-mediated cell death. Accordingly, the lung tissue sections from ABT-263 treated mice showed apicallyelevated apoptotic epithelial cells suggesting that epithelial extrusion or a programmed elimination might beinvolved in the removal of dying cells. In addition, the in-vitro studies with induced expression of Bik selectivelyremoved the dying cells by extrusion with a characteristic actinomyosin ring formation. Therefore, we proposeto investigate the cell death and extrusion process of airway epithelial cells by fixed- and live-cell imaging, andto interrogate the signaling pathways involved. We propose to test our findings in-vitro in both monolayer anddifferentiated culture settings, and in animal models. The efficacy of inhaled ABT-263 in regulating hyperplasticmucous cells will be tested in relevant mouse models of allergic asthma. More importantly, therapeutics forcontrolling mucous hypersecretion are limited with only few potential drugs currently in preclinical and clinicaltrials, therefore targeting hyperplastic mucous cells could represent a new class of mucolytic therapies. Theuse of ABT-263, an orally bioavailable Bcl-2 inhibitor that is currently being tested for human cancer treatmentcould help expedite its efficacy testing in asthmatics with debilitating mucous phenotype. In addition, theproposed studies will help develop a comprehensive understanding about the molecular mechanisms by whichblocking Bcl-2 facilitates the IL-13–mediated mucous cell death. The long term objective of these studies is tohelp understand the pathogenesis of mucus hypersecretion observed in chronic airway diseases.