Low Cellular NAD+ Compromises Lipopolysaccharide-Induced Inflammatory Responses via Inhibiting TLR4 Signal Transduction in Human Monocytes
Low Cellular NAD+ Compromises Lipopolysaccharide-Induced Inflammatory Responses via Inhibiting TLR4 Signal Transduction in Human Monocytes
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DOI：
10.4049/jimmunol.1801382
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发表时间：
2019-09-15
影响因子：
4.4
通讯作者：
Yndestad, Arne
中科院分区：
文献类型：
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作者：
Yang, Kuan;Lauritzen, Knut Huso;Yndestad, Arne
NAD(+) 是氧化还原代谢中重要的辅助因子，对代谢和炎症疾病有影响。然而，阐明 NAD(+) 对人原代单核细胞促炎特征的影响的数据很少。在本研究中，我们探讨了 NAD(+) 如何通过 PYD 结构域 3 (NLRP3) 炎症小体激活（两种关键的先天免疫反应）影响 TLR4 和 NOD 样受体。从健康个体的血沉棕黄层中分离出人原代单核细胞。细胞内 NAD(+) 由烟酰胺核苷和 NAMPT 抑制剂 FK866 控制。用 LPS 引发细胞，随后用 ATP 或胆固醇晶体激活或不激活 NLRP3，以分别分析 NAD(+) 水平对 TLR4 介导的 NF-κ B 激活和 NLRP3 活性的影响。对细胞因子释放进行定量，并通过蛋白质印迹和蛋白质组分析研究 TLR4 的下游信号通路。还探讨了 Sirtuin 和 PARP 抑制的影响。我们的主要发现是：1) 在体外暴露于 ATP 的 LPS 引发的人单核细胞中，NAD(+) 增加，IL-1 β 释放增加，2) FK866 消耗 NAD(+) 可以抑制 LPS 暴露的单核细胞中 NLRP3 依赖性和非依赖性炎症反应，3) 这种抑制不是由 Sirtuins 或 PARP1 的抑制引起的，4) 几种蛋白质 TLR4 信号通路的磷酸化是由 FK866 抑制的。受 FK866 介导的 NAD(+) 耗竭抑制，特别是 TAK1、IKK beta、IkB alpha、MEK 1/2、ERK 1/2 和 p38。因此，我们提出了一种 NAD(+) 影响 TLR4 信号转导的新机制。此外，我们的数据对之前关于 NAD(+) 与炎症之间相互作用的报告提出了质疑，并对烟酰胺核苷在炎症性疾病治疗中的使用提出了质疑。
NAD(+) is an essential cofactor in reduction-oxidation metabolism with impact on metabolic and inflammatory diseases. However, data elucidating the effects of NAD(+) on the proinflammatory features of human primary monocytes are scarce. In this study, we explored how NAD(+) affects TLR4 and NOD-like receptor with a PYD-domain 3 (NLRP3) inflammasome activation, two key innate immune responses. Human primary monocytes were isolated from buffy coats obtained from healthy individuals. Intracellular NAD(+) was manipulated by nicotinamide riboside and the NAMPT inhibitor FK866. Cells were primed with LPS with or without subsequent NLRP3 activation with ATP or cholesterol crystals to analyze the effects of NAD(+) levels on TLR4-mediated NF-kappa B activation and NLRP3 activity, respectively. Cytokine release was quantified, and the downstream signal pathway of TLR4 was investigated with Western blot and proteomic analysis. The impact of sirtuin and PARP inhibition was also explored. Our main findings were: 1) elevated NAD(+) enhanced IL-1 beta release in LPS-primed human monocytes exposed to ATP in vitro, 2) both NLRP3-dependent and -independent inflammatory responses in LPS-exposed monocytes were inhibited by NAD(+) depletion with FK866, 3) the inhibition was not caused by suppression of sirtuins or PARP1, and 4) phosphorylation of several proteins TLR4 signal pathway was inhibited by FK866-mediated NAD(+) depletion, specifically TAK1, IKK beta, IkB alpha, MEK 1/2, ERK 1/2, and p38. Hence, we suggest a novel mechanism in which NAD(+) affects TLR4 signal transduction. Furthermore, our data challenge previous reports of the interaction between NAD(+) and inflammation and question the use of nicotinamide riboside in the therapy of inflammatory disorders.