Edinburgh Research Explorer Macrophages exposed continuously to lipopolysaccharide and other agonists that act via toll-like receptors exhibit a sustained and additive activation state
Edinburgh Research Explorer Macrophages exposed continuously to lipopolysaccharide and other agonists that act via toll-like receptors exhibit a sustained and additive activation state
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背景资料：巨噬细胞通过激活Toll样受体家族的成员来感知微生物，Toll样受体家族启动与许多炎症相关基因转录相关的信号。在本文中，我们研究是否从Toll样受体[TLR]的信号是持续的，只要配体存在，以及是否对不同的TLR激动剂的反应是加性的。结果如下：用报告基因双重转染RAW 264巨噬细胞，其中IL-12 p40、ELAM或IL-6启动子控制萤火虫荧光素酶，而人IL-1 β启动子驱动海肾荧光素酶。所得的稳定系提供了通过TLR刺激物（包括LPS [TLR 4]、脂肽[TLR 2]和细菌DNA [TLR 9]）激活巨噬细胞的稳健测定，其中每个启动子展示其自身的内在特征。使用每种启动子，在8小时的时间内诱导荧光素酶活性，此后达到新的稳态。升高的表达需要激动剂的持续存在。对不同种类激动剂的持续反应是完全累加的。通过测量相同细胞中的诱导型细胞因子产生证实了这种模式。虽然TLR 4的同源二聚化介导对LPS的应答，但TLR 2似乎需要与另一种受体如TLR 6的异源二聚化。TLR 4或TLR 2加TLR 6的组成型活性形式的瞬时表达刺激IL-12启动子活性。LPS（一种TLR 4激动剂）的作用与TLR 2/6的作用是相加的，而与TLR 4的作用不是相加的，而脂肽（一种TLR 2激动剂）的作用与TLR 4的作用是相加的，而与TLR 2/6的作用不是相加的。细菌DNA的作用与TLR 4或TLR 2/6是相加的。结论：这些发现表明，通过任何一种TLR途径的最大激活并不排除通过另一种途径的进一步激活，这表明共同的下游调节组分是不受限制的。在暴露于TLR激动剂后，巨噬细胞进入持续激活状态，其中它们持续感知微生物挑战的存在。
Background: Macrophages sense microorganisms through activation of members of the Toll-like receptor family, which initiate signals linked to transcription of many inflammation associated genes. In this paper we examine whether the signal from Toll-like receptors [TLRs] is sustained for as long as the ligand is present, and whether responses to different TLR agonists are additive. Results: RAW264 macrophage cells were doubly-transfected with reporter genes in which the IL-12p40, ELAM or IL-6 promoter controls firefly luciferase, and the human IL-1 β promoter drives renilla luciferase. The resultant stable lines provide robust assays of macrophage activation by TLR stimuli including LPS [TLR4], lipopeptide [TLR2], and bacterial DNA [TLR9], with each promoter demonstrating its own intrinsic characteristics. With each of the promoters, luciferase activity was induced over an 8 hr period, and thereafter reached a new steady state. Elevated expression required the continued presence of agonist. Sustained responses to different classes of agonist were perfectly additive. This pattern was confirmed by measuring inducible cytokine production in the same cells. While homodimerization of TLR4 mediates responses to LPS, TLR2 appears to require heterodimerization with another receptor such as TLR6. Transient expression of constitutively active forms of TLR4 or TLR2 plus TLR6 stimulated IL-12 promoter activity. The effect of LPS, a TLR4 agonist, was additive with that of TLR2/6 but not TLR4, whilst that of lipopeptide, a TLR2 agonist, was additive with TLR4 but not TLR2/6. Actions of bacterial DNA were additive with either TLR4 or TLR2/6. Conclusions: These findings indicate that maximal activation by any one TLR pathway does not preclude further activation by another, suggesting that common downstream regulatory components are not limiting. Upon exposure to a TLR agonist, macrophages enter a state of sustained activation in which they continuously sense the presence of a microbial challenge.