CX3CR1 Is Expressed in Differentiated Human Ciliated Airway Cells and Co-Localizes With Respiratory Syncytial Virus on Cilia in a G Protein-Dependent Manner 

Kwang-Il Jeong  1 , Peter A Piepenhagen  2 , Michael Kishko  1 , Joshua M DiNapoli  1 , Rachel P Groppo  1 , Linong Zhang  1 , Jeffrey Almond  1 , Harry Kleanthous  1 , Simon Delagrave  1 , Mark Parrington  1             
Source: PLoS ONE
Publication Date: (2015)
Issue: 10: 6
Research Area:
Respiratory Research
Cells used in publication:
Epithelial, airway, human
Species: human
Tissue Origin: lung
Epithelial, Small Airway, human (SAEC)
Species: human
Tissue Origin: lung

Primary human small airway epithelial cells (SAEC; CC-2547S, Lonza, Walkersville, MD) of a healthy 11-year-old donor were grown and differentiated in a humidified atmosphere (5% CO2, 37°C) as described previously. Briefly, reaching 75–80% confluence in T75 tissue culture flasks fed with small airway growth medium (SAGM; CC-3281, Lonza) supplemented with growth factors and hormones (ALI SingleQuots Kit; CC-4538, Lonza), the cells were dissociated with trypsin/EDTA and seeded on the semipermeable membrane of transwell culture inserts (6.5 mm diameter, 0.4 µm pore size; Corning-Costar, Lowell, MA) coated with rat tail collagen type 1 (BD Biosciences, Bedford, MA). When confluence was reached, an air-liquid interface (ALI) was created to trigger differentiation by removing the growth medium from the apical compartment of the culture inserts set in 24-well plates and replacing growth medium in the basal compartment with differentiation medium (CC-3281, Lonza) supplemented with the differentiation inducer (ALI SingleQuots Kit; CC-4538, Lonza). Thereafter, the differentiation medium was changed in the basal compartment every other day, and the apical compartment was gently washed with the culture medium once or twice a week to remove accumulated debris and mucus. The cells fully differentiate in 21 to 25 days of ALI culture into ciliated cells, goblet cells, basal cells, and non-ciliated columnar cells.


Respiratory syncytial virus (RSV) is the principal cause of bronchiolitis in infants and a significant healthcare problem. The RSV Glycoprotein (G) mediates attachment of the virus to the cell membrane, which facilitates interaction of the RSV Fusion (F) protein with nucleolin, thereby triggering fusion of the viral and cellular membranes. However, a host protein ligand for G has not yet been identified. Here we show that CX3CR1 is expressed in the motile cilia of differentiated human airway epithelial (HAE) cells, and that CX3CR1 co-localizes with RSV particles. Upon infection, the distribution of CX3CR1 in these cells is significantly altered. Complete or partial deletion of RSV G results in viruses binding at least 72-fold less efficiently to cells, and reduces virus replication. Moreover, an antibody targeting an epitope near the G protein's CX3CR1-binding motif significantly inhibits binding of the virus to airway cells. Given previously published evidence of the interaction of G with CX3CR1 in human lymphocytes, these findings suggest a role for G in the interaction of RSV with ciliated lung cells. This interpretation is consistent with past studies showing a protective benefit in immunizing against G in animal models of RSV infection, and would support targeting the CX3CR1-G protein interaction for prophylaxis or therapy. CX3CR1 expression in lung epithelial cells may also have implications for other respiratory diseases such as asthma.