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./nMethods 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./nConclusions: 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.

中文翻译：

---

单核细胞通过旁分泌上调 TLR2 水平增强主动脉瓣间质细胞对 TLR2 刺激的炎症反应

背景和目的：与单核细胞浸润相关的慢性瓣膜炎症促进钙化性主动脉瓣疾病 (CAVD) 的进展。此外，由 Toll 样受体 (TLR) 介导的主动脉瓣间质细胞 (AVIC) 中的先天免疫上调细胞炎症、纤维化和成骨活性。目前，单核细胞和 AVICs 之间的促炎通讯及其潜在机制尚不清楚。我们假设单核细胞上调 AVIC 炎症活动。本研究旨在表征单核细胞和 AVIC 之间的相互作用，并阐明细胞间通讯的潜在机制。/n方法和结果：AVIC、单核细胞和共培养物暴露于低浓度的 TLR2 激活剂 Pam3CSK4 (0.03 µg/ml)。仅当与单核细胞共培养时，该剂量的 TLR2 激活剂才诱导 ICAM-1 和 VCAM-1 的 AVIC 产生显着增加。将来自 Pam3CSK4 处理的单核细胞（Pam3 CM，含有 0.1 µg/ml Pam3CSK4）的条件培养基添加到 AVIC 培养物（30% vol/vol；将 Pam3CSK4 稀释至 0.03 µg/ml）大大增加了粘附分子的表达，同时添加来自未经处理的单核细胞（对照CM）没有效果。AVIC 中 TLR2 的抑制或敲低显着降低了 Pam3 CM 诱导的 ICAM-1 和 VCAM-1 表达。此外，Pam3 CM 增加了 AVIC 中的 TLR2 水平。Pam3 CM 的多重 ELISA 分析确定了更高水平的 TNF-α。中和 TNF-α 消除了 Pam3 CM 对 AVIC TLR2 水平的影响，结论：本研究表明，活化的单核细胞使用旁分泌信号使 AVIC 对低水平 TLR2 激活剂的炎症反应敏感。致敏机制涉及来自单核细胞的 TNF-α 上调 AVIC TLR2 水平。主动脉瓣组织中浸润的单核细胞可能通过使 AVICs 对 TLR2 激活剂过敏而加剧瓣膜炎症。