LPS tolerance in macrophages. Mus musculus

Among the multiple mechanisms that control the intensity and duration of macrophage activation, the development of a state of refractoriness to a second stimulation in cells treated with LPS has long been recognized. Release of inhibitory cytokines and alterations in intracellular signaling pathways may be involved in the development of LPS tolerance. Although a number of molecules have been implicated, a detailed picture of the molecular changes in LPS tolerance is still missing. We have used a genome-wide gene expression analysis approach to (i) define which fraction of LPS target genes are subject to tolerance induction and (ii) identify genes that are expressed at high levels in tolerant macrophages. Our data show that in LPS tolerant macrophages the vast majority of LPS-induced gene expression is abrogated. The extent of tolerance induction varies for individual genes, and a small subset appears to be excepted. Compared to other negative control mechanisms of macrophages, e.g. IL-10-induced deactivation, LPS-tolerance inhibits a much wider range of transcriptional targets. Some previously described negative regulators of TLR-signaling (e.g. IRAK-M) were confirmed as expressed at higher levels in LPS-tolerant macrophages. In addition, we discuss other potential players in LPS tolerance identified in this group of genes. Keywords: pre and restimulation with LPS, tolerance induction Overall design: Cells were either prestimulated (1_X) or not (0_X) for 18h with 100 ng/ml LPS. Media was exchanged and after 2h rest, cells either restimulated (X_1) or not (X_0) with again 100 ng/ml LPS. This resulted in 4 experimental conditions: 0_0, 0_1, 1_0 and 1_1. There were 3 biological replicates done, resulting in 12 arrays altogether.

在调控巨噬细胞激活强度与持续时长的多种机制中，经脂多糖（LPS）处理的细胞对二次刺激产生无反应耐受状态的现象，早已被学界公认。抑制性细胞因子的释放以及细胞内信号通路的改变，可能参与了LPS耐受的发生发展。尽管已有诸多分子被关联至该过程，但LPS耐受背后完整的分子变化图景仍未明确。我们采用全基因组基因表达分析策略，旨在（i）明确脂多糖靶基因中可被诱导产生耐受的基因比例；（ii）筛选在耐受型巨噬细胞中高表达的基因。我们的数据显示，在LPS耐受的巨噬细胞中，绝大多数脂多糖诱导的基因表达均被阻断。不同基因的耐受诱导程度存在个体差异，仅有极小部分基因属于例外情况。相较于巨噬细胞的其他负调控机制（例如白细胞介素10（IL-10）介导的细胞失活），LPS耐受可抑制范围更广的转录靶标。部分此前已报道的Toll样受体（TLR）信号负调控因子（如白细胞介素1受体相关激酶M（IRAK-M））被证实可在LPS耐受巨噬细胞中呈高表达。此外，我们还对本研究基因集合中其他潜在的LPS耐受调控因子进行了讨论。关键词：脂多糖预刺激与二次刺激，耐受诱导 整体实验设计：细胞分别以100 ng/ml脂多糖预刺激18小时（标记为1_X）或不进行预刺激（标记为0_X）。更换培养基并静置2小时后，再以100 ng/ml脂多糖进行二次刺激（标记为X_1）或不进行二次刺激（标记为X_0），最终得到4组实验条件：0_0、0_1、1_0及1_1。本实验设置3次生物学重复，总计获得12张基因表达阵列数据。