A WNT4- and DKK3-driven canonical to noncanonical Wnt signaling switch controls multiciliogenesis.

Multiciliated cells contain hundreds of cilia whose directional movement powers the mucociliary clearance of the airways, a vital host defense mechanism. Multiciliated cell specification requires canonical Wnt signaling, which then must be turned off. Next, ciliogenesis and polarized ciliary orientation are regulated by noncanonical Wnt/planar cell polarity (Wnt/PCP) signaling. The mechanistic relationship between the Wnt pathways is unknown. We show that DKK3, a secreted canonical Wnt regulator and WNT4, a noncanonical Wnt ligand act together to facilitate a canonical to noncanonical Wnt signaling switch during multiciliated cell formation. In primary human airway epithelial cells, DKK3 and WNT4 CRISPR knockout blocks, whereas ectopic expression promotes, multiciliated cell formation by inhibiting canonical Wnt signaling. Wnt4 and Dkk3 single-knockout mice also display defective ciliated cells. DKK3 and WNT4 are co-secreted from basal stem cells and act directly on multiciliated cells via KREMEN1 and FZD6, respectively. We provide a novel mechanism that links specification to cilium biogenesis and polarization for proper multiciliated cell formation.

Notch signaling inactivation by small molecule γ-secretase inhibitors restores the multiciliated cell population in the airway epithelium.

Multiciliated cell loss is a hallmark of airway epithelial remodeling in chronic inflammatory airway diseases including cystic fibrosis (CF), asthma, and chronic obstructive pulmonary disease. It disrupts mucociliary clearance, which fuels disease progression. Effective clearance requires an optimal proportion of multiciliated and secretory cells. This is controlled by Notch signaling such that between two adjacent cells the one that activates Notch becomes a secretory cell and the one that avoids Notch activation becomes a multiciliated cell. Consequently, blocking Notch by a small molecule inhibitor of the γ-secretase enzyme that cleaves the Notch receptor for signal activation directs differentiation toward the multiciliated lineage. Thus, γ-secretase inhibitor (GSI) treatment may alleviate multiciliated cell loss in lung disease. Here, we demonstrate the therapeutic restoration of multiciliated cells by the GSI LY450139 (semagacestat). LY450139 increased multiciliated cell numbers in a dose-dependent manner in healthy primary human nasal epithelial cells (HNECs) during differentiation and in mature cultures, but not when applied during early epithelialization of progenitors. LY450139 did not impact stem cell proliferation. Basal and apical administration were equally effective. In healthy adult mice, LY450139 increased multiciliated cell numbers without detectible toxicity. LY450139 also increased multiciliated cells and decreased excess mucus secretory cells in CF HNECs and IL-13 remodeled healthy HNECs. LY450139 normalized multiciliated cell numbers in CF HNECs without interfering with the activity of CFTR modulator compounds. In summary, we demonstrate that GSI administration is a promising therapeutic to restore multiciliated cells and potentially improve epithelial function in a wide range of chronic lung diseases.NEW & NOTEWORTHY Our findings show that low-dose, short-term topical or systemic γ-secretase inhibitor treatment may lead to restoration of multiciliated cells without toxicity and potentially improve epithelial function in a wide range of chronic lung diseases.

Anionic Pulmonary Surfactant Lipid Treatment Inhibits Rhinovirus A Infection of the Human Airway Epithelium.

Rhinoviruses (RVs) are major instigators of acute exacerbations of asthma, COPD, and other respiratory diseases. RVs are categorized into three species (RV-A, RV-B, and RV-C), which comprise more than 160 serotypes, making it difficult to develop an effective vaccine. Currently, no effective treatment for RV infection is available. Pulmonary surfactant is an extracellular complex of lipids and proteins that plays a central role in regulating innate immunity in the lung. The minor pulmonary surfactant lipids, palmitoyl-oleoyl-phosphatidylglycerol (POPG) and phosphatidylinositol (PI), are potent regulators of inflammatory processes and exert antiviral activity against respiratory syncytial virus (RSV) and influenza A viruses (IAV). In the current study, we examined the potencies of POPG and PI against rhinovirus A16 (RV-A16) in primary human airway epithelial cells (AECs) differentiated at an air-liquid interface (ALI). After AECs were infected with RV-A16, PI reduced the viral RNA copy number by 70% and downregulated (55-75%) the expression of antiviral (MDA5, IRF7, and IFN-lambda) and CXCL11 chemokine genes. In contrast, POPG only slightly decreased MDA5 (24%) and IRF7 (11%) gene expression but did not inhibit IFN-lambda gene expression or RV-A16 replication in AECs. However, both POPG and PI inhibited (50-80%) IL6 gene expression and protein secretion and CXCL11 protein secretion. PI treatment dramatically attenuated global gene expression changes induced by RV-A16 infection alone in AECs. The observed inhibitory effects were indirect and resulted mainly from the inhibition of virus replication. Cell-type enrichment analysis of viral-regulated genes opposed by PI treatment revealed the PI-inhibited viral induction of goblet cell metaplasia and the virus-induced downregulation of ciliated, club, and ionocyte cell types. Notably, the PI treatment also altered the ability of RV-A16 to regulate the expression of some phosphatidylinositol 4-kinase (PI4K); acyl-CoA-binding, domain-containing (ACBD); and low-density lipoprotein receptor (LDLR) genes that play critical roles in the formation and functioning of replication organelles (ROs) required for RV replication in host cells. These data suggest PI can be used as a potent, non-toxic, antiviral agent for RV infection prophylaxis and treatment.