Arranz Alicia, Androulidaki Ariadne, Zacharioudaki Vassiliki, Martinez Carmen, Margioris Andrew N, Gomariz Rosa P, Tsatsanis Christos
Department of Clinical Chemistry-Biochemistry, School of Medicine, University of Crete, Heraklion GR-710 03, Crete, Greece.
Mol Immunol. 2008 May;45(10):2970-80. doi: 10.1016/j.molimm.2008.01.023. Epub 2008 Mar 11.
Toll-like receptor 4 (TLR4) recognizes and initiates signals from Gram-negative bacterial lipopolysaccharide (LPS) triggering the inflammatory response. Expression levels of TLR4 on macrophages partly regulate the magnitude of the response to LPS. Vasoactive Intestinal Peptide (VIP) is known to block inflammatory responses by inhibiting pro-inflammatory cytokine production from activated macrophages. In the present report we demonstrate that VIP directly suppressed TLR4 expression on naïve primary mouse macrophages utilizing signalling cascades that control TLR4 transcription. VIP-induced suppression of TLR4 occurred at the transcriptional level by decreasing PU.1 DNA binding. Mutation of the proximal PU.1 but not the AP-1-binding site on the TLR4 promoter abrogated VIP-induced suppression of TLR4 transcription. Moreover, inhibition of PI3K by wortmannin or homologous deletion of the Akt1 isoform, a pathway known to act as a negative regulator of macrophage activation, alleviated the suppressive action of VIP on TLR4 expression. To evaluate the biological significance of VIP effect on TLR4 expression, Raw264.7 macrophages were pre-treated with VIP for 24h and then exposed to LPS. Pre-treatment with VIP rendered macrophages hypo-responsive to LPS resulting in reduced pro-inflammatory cytokine production. Moreover, in vivo administration of VIP in C57BL/6 mice resulted in lower IL-6 production upon treatment with LPS. Overall, the results indicate that VIP promotes endotoxin tolerance by downregulating TLR4 expression via Akt1.
Toll样受体4（TLR4）识别革兰氏阴性菌脂多糖（LPS）并启动信号，触发炎症反应。巨噬细胞上TLR4的表达水平部分调节对LPS反应的强度。已知血管活性肠肽（VIP）通过抑制活化巨噬细胞产生促炎细胞因子来阻断炎症反应。在本报告中，我们证明VIP利用控制TLR4转录的信号级联直接抑制未活化的原代小鼠巨噬细胞上的TLR4表达。VIP诱导的TLR4抑制通过降低PU.1与DNA的结合在转录水平上发生。TLR4启动子近端PU.1位点而非AP-1结合位点的突变消除了VIP诱导的TLR4转录抑制。此外，渥曼青霉素抑制PI3K或Akt1亚型的同源缺失（已知该途径作为巨噬细胞活化的负调节因子）减轻了VIP对TLR4表达的抑制作用。为了评估VIP对TLR4表达影响的生物学意义，将Raw264.7巨噬细胞用VIP预处理24小时，然后暴露于LPS。用VIP预处理使巨噬细胞对LPS反应性降低，导致促炎细胞因子产生减少。此外，在C57BL/6小鼠体内给予VIP导致用LPS处理后IL-6产生降低。总体而言，结果表明VIP通过Akt1下调TLR4表达来促进内毒素耐受性。
