Monocytes enhance the inflammatory response to TLR2 stimulation in aortic valve interstitial cells through paracrine up-regulation of TLR2 level

Background and Objectives: Chronic valvular inflammation associated with monocyte infiltration promotes calcific aortic valve disease (CAVD) progression. Further, innate immunity in aortic valve interstitial cells (AVICs), mediated by Toll-like receptors (TLRs), up-regulates cellular inflammatory, fibrogenic and osteogenic activities. Currently, the pro-inflammatory communication between monocytes and AVICs and the underlying mechanism are unclear. We hypothesized that monocytes up-regulate AVIC inflammatory activity. This study sought to characterize the interaction between monocytes and AVICs and to elucidate the mechanism underlying cell-to-cell communication. Methods and Results: AVICs, monocytes and co-cultures were exposed to a low concentration of TLR2 activator Pam3CSK4 (0.03 µg/ml). The TLR2 activator at this dose induced a marked increase in AVIC production of ICAM-1 and VCAM-1 only when co-cultured with monocytes. Adding conditioned medium from Pam3CSK4-treated monocytes (Pam3 CM, containing 0.1 µg/ml of Pam3CSK4) to AVIC culture (30% vol/vol; diluting Pam3CSK4 to 0.03 µg/ml) greatly increased the expression of adhesion molecules while adding conditioned medium from untreated monocytes (control CM) had no effect. Inhibition or knockdown of TLR2 in AVICs markedly reduced ICAM-1 and VCAM-1 expression induced by Pam3 CM. Further, Pam3 CM increased TLR2 levels in AVICs. Multiplex-ELISA analysis of Pam3 CM identified greater levels of TNF-α. Neutralization of TNF-α abolished the effect of Pam3 CM on AVIC TLR2 levels, resulting in marked attenuation of its potency in the induction of adhesion molecule expression. Conclusions: This study demonstrates that activated monocytes use paracrine signaling to sensitize AVICs for inflammatory responses to a low level of TLR2 activator. The mechanism of sensitization involves up-regulation of AVIC TLR2 levels by TNF-α from monocytes. Infiltrated monocytes in aortic valve tissue may exacerbate valvular inflammation by rendering AVICs hypersensitive to TLR2 activators.