Department of Molecular Microbiology & Immunology, Oregon Health & Science University, Portland, 97239, USA.
J Neuroinflammation. 2011 Oct 14;8:140. doi: 10.1186/1742-2094-8-140.
Toll-like receptor 4 (TLR4) is activated in response to cerebral ischemia leading to substantial brain damage. In contrast, mild activation of TLR4 by preconditioning with low dose exposure to lipopolysaccharide (LPS) prior to cerebral ischemia dramatically improves outcome by reprogramming the signaling response to injury. This suggests that TLR4 signaling can be altered to induce an endogenously neuroprotective phenotype. However, the TLR4 signaling events involved in this neuroprotective response are poorly understood. Here we define several molecular mediators of the primary signaling cascades induced by LPS preconditioning that give rise to the reprogrammed response to cerebral ischemia and confer the neuroprotective phenotype.
C57BL6 mice were preconditioned with low dose LPS prior to transient middle cerebral artery occlusion (MCAO). Cortical tissue and blood were collected following MCAO. Microarray and qtPCR were performed to analyze gene expression associated with TLR4 signaling. EMSA and DNA binding ELISA were used to evaluate NFκB and IRF3 activity. Protein expression was determined using Western blot or ELISA. MyD88-/- and TRIF-/- mice were utilized to evaluate signaling in LPS preconditioning-induced neuroprotection.
Gene expression analyses revealed that LPS preconditioning resulted in a marked upregulation of anti-inflammatory/type I IFN-associated genes following ischemia while pro-inflammatory genes induced following ischemia were present but not differentially modulated by LPS. Interestingly, although expression of pro-inflammatory genes was observed, there was decreased activity of NFκB p65 and increased presence of NFκB inhibitors, including Ship1, Tollip, and p105, in LPS-preconditioned mice following stroke. In contrast, IRF3 activity was enhanced in LPS-preconditioned mice following stroke. TRIF and MyD88 deficient mice revealed that neuroprotection induced by LPS depends on TLR4 signaling via TRIF, which activates IRF3, but does not depend on MyD88 signaling.
Our results characterize several critical mediators of the TLR4 signaling events associated with neuroprotection. LPS preconditioning redirects TLR4 signaling in response to stroke through suppression of NFκB activity, enhanced IRF3 activity, and increased anti-inflammatory/type I IFN gene expression. Interestingly, this protective phenotype does not require the suppression of pro-inflammatory mediators. Furthermore, our results highlight a critical role for TRIF-IRF3 signaling as the governing mechanism in the neuroprotective response to stroke.
Toll 样受体 4（TLR4）在脑缺血后被激活，导致大量脑损伤。相比之下，在脑缺血前用低剂量脂多糖（LPS）预处理轻度激活 TLR4，通过重新编程损伤后的信号反应，显著改善了预后。这表明 TLR4 信号可以被改变以诱导内源性神经保护表型。然而，在这种神经保护反应中涉及的 TLR4 信号事件知之甚少。在这里，我们定义了 LPS 预处理诱导的初级信号级联反应中的几个分子介质，这些介质导致对脑缺血的重新编程反应，并赋予神经保护表型。
C57BL6 小鼠在短暂性大脑中动脉闭塞（MCAO）前用低剂量 LPS 预处理。MCAO 后采集皮质组织和血液。进行微阵列和 qtPCR 分析以分析与 TLR4 信号相关的基因表达。EMSA 和 DNA 结合 ELISA 用于评估 NFκB 和 IRF3 活性。使用 Western blot 或 ELISA 测定蛋白表达。利用 MyD88-/-和 TRIF-/-小鼠来评估 LPS 预处理诱导的神经保护中的信号转导。
基因表达分析表明，LPS 预处理导致缺血后抗炎/Ⅰ型 IFN 相关基因的显著上调，而缺血后诱导的促炎基因虽然存在，但不受 LPS 差异调节。有趣的是，尽管观察到促炎基因的表达，但在 LPS 预处理的小鼠中风后，NFκB p65 的活性降低，NFκB 抑制剂，包括 Ship1、Tollip 和 p105 的存在增加。相比之下，IRF3 活性在 LPS 预处理的小鼠中风后增强。TRIF 和 MyD88 缺陷型小鼠表明，LPS 诱导的神经保护依赖于 TLR4 信号通过 TRIF，其激活 IRF3，但不依赖于 MyD88 信号。
我们的研究结果描述了与神经保护相关的 TLR4 信号事件的几个关键介质。LPS 预处理通过抑制 NFκB 活性、增强 IRF3 活性和增加抗炎/Ⅰ型 IFN 基因表达，重新定向 TLR4 信号对中风的反应。有趣的是，这种保护表型不需要抑制促炎介质。此外，我们的结果突出了 TRIF-IRF3 信号作为中风后神经保护反应的控制机制的关键作用。
