B76-07 Overproduction of Muc5b Impairs the Mucociliary Transport in Alpha 1 Antitrypsin Deficiency

Abstract Rationale Alpha-1-antitrypsin deficiency (AATD) is a genetic risk factor that accelerates the onset of chronic obstructive pulmonary disease in smokers, impairing mucociliary clearance (MCC). AATD disrupts the protease-antiprotease balance, promoting epithelial injury and mucus overproduction through cytokine and neutrophil elastase (NE) driven feedback, thereby worsening small airway obstruction. Mechanisms by which NE in AATD contributes to mucus stasis in the small airways are unknown. Here, we hypothesize that Muc5b overexpression contributes to the differentiation of club cells to goblet cells in AATD by enhanced NE activity. To investigate the role of Muc5b in AATD-related mucociliary dysfunction, we examined its association in human and ferret bronchial epithelial cells (BECs) exposed to cigarette smoke extract (CSE), following targeted Muc5b inhibition and treatment with CSE, NE, or both. Methods First, we performed single-cell RNA-sequencing (scRNA-seq) of primary BECs derived from an AATD patient treated with 1% CSE vs. vehicle control. To determine whether uncontrolled NE activity in AATD worsens CSE-induced mucociliary dysfunction via Muc5B regulation, we silenced MUC5B using siRNA and treated cells with NE, CSE, or both. Knockdown was confirmed by RT-PCR and immunofluorescence (IF), and MCC was assessed using micro-optical coherence tomography (µOCT) imaging. Ongoing experiments involve Muc5B overexpression using CRISPR activation with a lentiviral dCas9-VPR system. Gene expression is being evaluated by IF and Western blotting to determine whether Muc5b knockdown or overexpression influences MCC in AATD and potentiates the effects of CSE. Cytokine will also be measured for the inflammatory response. Results scRNA-seq and IF exhibited an increased Muc5B-positive goblet cell population following CSE exposure compared to vehicle controls. Primary HBE treated with NE upregulated Muc5b (1.8 fold, p = 0.01), and the effect was further enhanced by CSE treatment after NE treatment (2.4 fold, p = 0.01). μOCT analysis of CSE-exposed AATD-derived HBEs demonstrated a decline in MCT following CSE treatment (15% reduction; p = 0.001, N = 3). A further decrease in MCT was observed with combined NE (1μM) and 1% CSE treatment vs vehicle (20% reduction, p = 0.001), indicating an enhanced impairment of mucociliary function. Further, intervention with Muc5b-specific siRNA reduced expression of Muc5b (90%, p = 0.001, N = 3). Neither CSE, NE, nor a combination induced Muc5b expression with Muc5b knockdown. Conclusion We demonstrate that Muc5b overproduction worsens MCT in AATD by NE and exacerbates the effect of smoke exposure; the effect can be attenuated by siRNA Muc5b knockdown. Findings identify Muc5b as a potential therapeutic target for mitigating AATD-associated airway obstruction. This abstract is funded by: Alpha 1 foundation, American lung association, ATS, DREAM council, CF foundation