The combination of TLR 3 and 7/8 agonists, and RNA extracted from RV-induced conditioned medium or RV stock caused PGE2 induction and β2 adrenoceptor desensitization on ASMCs, but not RNA extracted from HBECs.
Control and RV-induced conditioned medium was generated from HBEC (n = 2) and pooled. ASMCs (n = 6) were treated with this pooled control or RV-induced conditioned medium; or untreated (BEGM), poly I:C (50 µg/mL), imiquimod (30 µg/mL) and poly I:C & imiquimod (50 µg/mL, 30 µg/mL respectively) in BEGM or in the presence of the control conditioned medium for 3 days (A, B). (Figure C-F) Control and RV-induced conditioned medium was generated from HBEC (n = 3) and pooled. Total RNA was extracted from: control- (53.91 ng/µL), RV-induced conditioned medium (300.25 ng/µL), RV stock (567.35 ng/µL) and cell lysate collected from a sub-confluent 75 cm2 flask of HBEC (300 ng/µL) using a miRNeasy Mini purification kit and amount of RNA quantified using a spectrophotometer. ASMCs (n = 6) were treated with pooled control conditioned medium (control), or total extracted RNA collected from those sources in the presence of control conditioned medium for 3 days. PGE2 was measured using an ELISA (A, C) and isoprenaline induced cAMP was measured using a cAMP functional assay (B, D-F). Data represent mean ± SEM. Statistical differences were detected using 1-way ANOVA with Bonferroni post test comparisons to respective BEGM, control conditioned medium only (Control) or Poly I:C *p<0.05, #p<0.05.