Abstract Rationale Mucus stasis is a hallmark of muco-obstructive airway diseases (MOADs), including cystic fibrosis (CF), leading to airway obstruction and impaired lung function. Recent advances in CF therapies have focused on correcting the underlying CFTR defect through modulator therapy, improving both mucus transport and lung function in these patients. However, these therapies do not correct the underlying mucus defect, making it unavailable for non-modulator responsive patients with CF and other MOADs. Mucus is comprised of mucins, large o-glycoproteins that are extensively sialylated. Sialic acids impart negative charge and influence mucin interactions with the ionic environment, yet their role in mucus pathology remains underexplored. We look to evaluate sialylation in the CF airway and determine its regulation of mucin structure and mucus function. Methods We analyzed single-cell transcriptomic data (GSE150674) from CF and non-CF bronchial epithelial cells using co-expression gene set enrichment focused on sialylation-related glycogenes (i.e. glyco-enzymes, lectins, sugar-nucleotide synthesis). Area Under the Curve analysis quantified gene set enrichment across cell types and diagnoses. Bronchial epithelial cells (BECs) from CF and non-CF patients were cultured at air-liquid interface (ALI), and pooled mucus was analyzed for sialylation. In-vitro, healthy, salivary MUC5B was treated with exogenous sialidase (V. cholera) to reduce sialylation, and glycoform abundance were assessed via agarose-polyacrylamide gel electrophoresis (Ag-PAGE). Rate-zonal centrifugation (RZC) was used to separate slow- and fast-sedimenting MUC5B, and sialylation levels were assessed using Ag-PAGE blotting. To reduce sialylation in-vivo, excised trachea from WT rats treated with sialidase, sialyltransferase inhibitor, were evaluated for mucociliary transport (MCT) and air-surface liquid (ASL) depth via micro-ocular coherence tomography (µOCT). WT and Cftr-/- rat tracheae were similarly evaluated. Results CF epithelial cells exhibited elevated sialylation scores, or differential co-expression of sialo-glycogenes, across major cell types (e.g. Ciliated, Basal, and Secretory). In-vitro, mucus collected from CF BECs showed reduced sialylation compared to non-CF controls, suggesting this altered sialo-glycogene expression in CF may be reducing sialylation. Sialidase treatment of MUC5B reproduced a pathological glycoform seen in MOADs. Moreover, slow-sedimenting (linear) MUC5B polymers showed higher sialylation than fast-sedimenting (compact) forms, correlating reduced sialylation with mucin compaction. In rat tracheae, reduction of sialylation - through sialidase treatment or sialyltransferase inhibition - impaired MCT, mirroring CF pathology. Conclusions Sialylation plays a critical role in mucin structure and function. Its dysregulation may contribute to mucus pathology in CF and other MOADs. Increasing mucin sialylation could represent a novel therapeutic strategy to alleviate the burden of obstructive mucus. This abstract is funded by: T32GM146611-02 to HJM; CF Foundation 008316G224 to JWB
McIntire-Ray et al. (Fri,) studied this question.