TLR3 activation stimulates TLR2 expression and innate immune responses to TLR2 ligands in airway epithelial cells

Toll‐like receptor (TLR1‐6) mRNAs are expressed in normal human bronchial epithelial (NHBE) cells with higher basal levels of TLR3. Expression of TLR2 mRNA was enhanced by treatment with Poly IC, a TLR3 ligand but not by the TLR2 ligand, PAM3CSK4, suggesting that viral infection can alter epithelial sensitivity and response to microbial components sensed by TLR2. Western blot analysis revealed that Poly IC pretreatment (24 hrs) produced up‐regulation of TLR2 protein. Activation of TLR3 also up‐regulated mRNA expression of MyD88 and TIRAP, adaptor molecules that are involved in TLR2 signaling. To assess consequences of TLR3 activation on TLR2 function, monolayers of NHBE cells were pretreated with Poly IC for 24 hrs and then stimulated with PAM3CSK4 for another 24 hrs. ELISA analysis of media harvested from the apical and basolateral compartments revealed significantly higher secretion of IL‐6 and GM‐CSF after Poly IC stimulation compared with PAM3CSK4 or flagellin (TLR5 ligand) and that apical secretion was significantly greater than basolateral secretion. Additionally, PAM3CSK4 stimulation of IL‐6 and GM‐CSF secretion following Poly IC pretreatment was significantly greater than the effects of PAM3CSK4 without Poly IC pretreatment. Our findings indicate that TLR3 activation enhances expression of the TLR2 signaling pathway and potentiates the functional response of the airway epithelium to TLR2 ligands.