University of Salerno, Department of Pharmacy, DIFARMA, Fisciano, Italy,
University of Salerno, Department of Pharmacy, DIFARMA, Fisciano, Italy.
Cell Physiol Biochem. 2021 Apr 30;55(2):222-234. doi: 10.33594/000000361.
BACKGROUND/AIMS: Sphingosine-1-phosphate (S1P) is a membrane-derived bioactive phospholipid involved in many lung physiological and pathological processes. Higher levels of S1P have been registered in a broad range of respiratory diseases, including inflammatory disorders and cancer. The aim of our study was to understand the role of S1P in healthy versus tumor cells after Toll-Like Receptors (TLRs) activation, well-known modulators of sphingolipid metabolism.
Lung adenocarcinoma cells and non-pathological human fibroblasts were stimulated with unmethylated Cytosine phosphate Guanosine (CpG), the TLR9 ligand, and S1P-dependent TNF-α release was evaluated by means of ELISA. Immunofluorescence and LC-MS/MS analysis were performed to evaluate/quantify S1P generation following TLR9 activation.
We found that S1P was involved in TLR9-induced TNF-α release in that the inhibition of both ceramidase and sphingosine kinase I/II (SPHK I/II) significantly reduced the levels of TNF-α after TLR9 triggering in lung adenocarcinoma cells. These results were not observed in healthy fibroblasts, implying that this pathway was mainly involved in pathological conditions. Moreover, the activation of TLR4 by means of LPS did not have similar effects as in the case of CpG-stimulated TLR9. Importantly, the activation of TLR9 induced S1P generation and allowed it to interact on the outside membrane receptor S1P and S1P via the efflux through its membrane transporter SPNS2. Indeed, both the blockade of S1P and the transporter SPNS2 significantly reduced the activity of S1P on TNF-α release from lung adenocarcinoma cells.
Our study identifies a novel inflammatory pathway in that TLR9 increases the pro-inflammatory cytokine release, such as TNF-α, via the induction of a ceramide/S1P imbalance in favor of S1P, adding a novel puzzle piece in TLR9-orchestrated inflammatory pathway and shedding more light on the role of the higher levels of S1P during inflammatory conditions.
背景/目的：鞘氨醇-1-磷酸（S1P）是一种膜衍生的生物活性磷脂，参与许多肺部生理和病理过程。在广泛的呼吸系统疾病中，包括炎症性疾病和癌症，都检测到较高水平的 S1P。我们的研究目的是了解 Toll 样受体（TLR）激活后 S1P 在健康细胞与肿瘤细胞中的作用，TLR 是鞘脂代谢的已知调节剂。
用未甲基化的胞嘧啶磷酸鸟嘌呤（CpG）刺激肺腺癌细胞和非病理人成纤维细胞，并通过 ELISA 评估 S1P 依赖性 TNF-α 释放。通过免疫荧光和 LC-MS/MS 分析评估/定量 TLR9 激活后 S1P 的产生。
我们发现 S1P 参与 TLR9 诱导的 TNF-α 释放，因为在 TLR9 触发后，鞘氨醇酶和鞘氨醇激酶 I/II（SPHK I/II）的抑制均显著降低了肺腺癌细胞中 TNF-α 的水平。在健康成纤维细胞中未观察到这些结果，这表明该途径主要涉及病理条件。此外，用 LPS 激活 TLR4 没有像 CpG 刺激 TLR9 那样的类似作用。重要的是，TLR9 的激活诱导 S1P 的产生，并允许其通过其膜转运蛋白 SPNS2 从外面膜受体 S1P 和 S1P 外排。事实上，阻断 S1P 和转运蛋白 SPNS2 都显著降低了 S1P 对肺腺癌细胞中 TNF-α 释放的活性。
我们的研究确定了一种新的炎症途径，即 TLR9 通过诱导有利于 S1P 的神经酰胺/S1P 失衡来增加促炎细胞因子（如 TNF-α）的释放，为 TLR9 协调的炎症途径增加了一个新的拼图，并且在炎症条件下 S1P 水平升高的作用更加清晰。
