(449) Pathways Balancing Basal Mucin and CFTR-mediated Fluid Secretion in the Human Small Airway

Thursday, November 3, 2022

12:45 PM – 1:25 PM ET

Presenting Author(s)

Kenichi Okuda, MD, PhD

Assistant Professor
Marsico Lung Institute/Cystic Fibrosis Research Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
Chapel Hill, North Carolina, United States

Co-Author(s)

Satoko Nakano

Research Technician
Marsico Lung Institute/Cystic Fibrosis Research Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
Chapel Hill, North Carolina, United States
AS

Avishi Singh

Marsico Lung Institute/Cystic Fibrosis Research Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA, United States
HD

Hong Dang

Bioinformatician
Marsico Lung Institute/Cystic Fibrosis Research Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA, United States
LM

Lisa C. Morton

Research Specialist
Marsico Lung Institute/Cystic Fibrosis Research Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA, United States
RG

Rodney C. Gilmore

Research Specialist
Marsico Lung Institute/Cystic Fibrosis Research Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA, United States
Rhianna E. Lee

PhD Student
Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA, United States
SG

Samuel C. Gallant

Research Technician
Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA, United States
NQ

Nancy L. Quinney

Research Specialist
Marsico Lung Institute/Cystic Fibrosis Research Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
Chapel Hill, North Carolina, United States
Deborah M. Cholon, PhD (she/her/hers)

Research Associate
Marsico Lung Institute/Cystic Fibrosis Research Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
Chapel Hill, North Carolina, United States
Martina Gentzsch, PhD (she/her/hers)

Associate Professor
Marsico Lung Institute and Cystic Fibrosis Research Center, Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
Chapel Hill, North Carolina, United States
SR

Scott H. Randell

Professor
Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA, United States
WO

Wanda K. O'Neal

United States
RB

Richard C. Boucher

Director
Marsico Lung Institute/Cystic Fibrosis Research Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA, United States

Background: Mucociliary clearance is heavily affected by mucus concentration with its attendant biophysical properties. Mucus concentration is tightly regulated by luminal mucin secretion and mucus hydration. While small (distal) airways (< 2 mm in diameter) constitute the earliest, most affected region in CF, how mucus concentrations are regulated in the small airway region remains unknown. Our prior studies have demonstrated small airway superficial epithelia are: 1) a major source of MUC5B production in the lung; and 2) a site of robust CFTR-mediated fluid secretion. These studies also localized both MUC5B secretion and CFTR-mediated ion transport activities to small airway secretory cells. Here, we sought to identify pathways balancing basal mucin and CFTR-mediated fluid secretion in small airway secretory cells.

Methods: Single cell RNA-seq (scRNA-seq) was performed on matched large and small airway epithelial (LAE and SAE, respectively) cultures (N=3) to transcriptionally characterize region-specific airway epithelial cell types. A pathway enriched in CFTR+/MUC5B+ small airway secretory cells was pharmacologically inhibited to test whether it regulated basal mucin secretion and CFTR function in SAE cells. A lentiviral vector that selectively transduced the wild-type (WT) CFTR gene into CF SAE secretory cells under the control of the rat club cell specific protein (rCCSP) promoter was used to test the competency of small airway secretory cells for CFTR function restoration. CF SAE data were compared to matched CF LAE data.

Results: A total of 24,170 cells (LAE 49%, SAE 51%) were analyzed by scRNA-seq. scRNA-seq identified an independent airway secretory cell subset predominantly comprised of SAE cells characterized by: 1) high levels of small airway marker gene expression (SFTPB, SCGB3A2); 2) high CFTR and MUC5B expression; 3) high expression of the IL-1 receptor gene (IL1R1); and 4) upregulation of downstream genes of IL-1/NF-κB signaling pathways. To test whether basal mucin (MUC5B) and CFTR-mediated fluid secretion is regulated via endogenous IL-1/NF-κB signaling in small airway secretory cells, ion transport function and MUC5B secretion rates were measured in SAE cultures where IL-1 signaling was inhibited by IL-1 receptor antagonist (IL1Ra) administration. Baseline expression of IL-1/NF-κBdownstream genes was suppressed in IL1Ra treated SAE cells. Notably, baseline MUC5B secretion rates and CFTR-mediated Cl^-secretion were also reduced in IL1Ra treated SAE cells. Utilizing the lentiviral vector, the WT CFTR gene was transduced via the rCCSP promoter in CF SAE and LAE cells. Whole mount immunohistochemistry revealed colocalization of CCSP and CFTR protein in non-ciliated cells in rCCSP-CFTR-transduced CF cultures. rCCSP-CFTR-transduced CF LAE and SAE cultures exhibited WT CFTR protein expression with restored CFTR-mediated Cl^-secretion in response to forskolin. Importantly, rCCSP-CFTR-transduced CF SAE exhibited greater restoration of CFTR function than LAE cultures.

Conclusions: A unique small airway secretory cell regulates basal mucin and CFTR-mediated fluid secretion at least partially by endogenous IL-1/NF-κB pathways in human distal airways. This unique small airway secretory cell is a therapeutic target for molecular therapy for CFTR restoration.

Acknowledgements: This work was supported by NIH (DK065988, P30DK065988) and CFF (OKUDA20G0, BOUCHER19XX0, and BOUCHE15R0).

References: