Anti-microbial immunity is impaired in COPD patients with frequent exacerbations

Aran Singanayagam; Su-Ling Loo; Maria Calderazzo; Lydia J Finney; Maria-Belen Trujillo Torralbo; Eteri Bakhsoliani; Jason Girkin; Punnam Veerati; Prabuddha S Pathinayake; Kristy S Nichol; Andrew Reid; Joseph Foottit; Sebastian L Johnston; Nathan W Bartlett; Patrick Mallia

Primary BECs obtained bronchoscopically from patients with COPD (GOLD Stage II or III) were grown until confluent and differentiated at air liquid-interface (ALI), as previously described [19] [20] [21] . All subjects included gave informed written consent and the study protocol was approved by the Hunter New England Human Research Ethics Committee (05/08/10/3.09). Primary cells were grown in complete bronchial epithelial cell growth medium (BEGM)(Lonza) with growth factor supplements in submerged monolayer culture and then seeded at 2 x 10 5 cells/mL in transwells until confluent. RV-A1 infection (MOI 0.1) was applied to the apical surface for 2 hours in 250μl bronchial epithelial basal medium (BEBM) minimal at 35 o C. Following this, infection media was replaced with 500μl fresh BEBM minimal for the remainder of the experiment. Samples were collected at 72 hours post-infection with apical media removed and stored for protein expression analyses. Type III IFN proteins were measured using a custom-designed LEGENDplex kit (BioLegend). IFN-β was measured using the Verikine ELISA (PBL).