DataSheet_1_Intracellular NAD+ Depletion Confers a Priming Signal for NLRP3 Inflammasome Activation.pdf

Nicotinamide adenine dinucleotide (NAD+) is an important cofactor in many redox and non-redox NAD+-consuming enzyme reactions. Intracellular NAD+ level steadily declines with age, but its role in the innate immune potential of myeloid cells remains elusive. In this study, we explored whether NAD+ depletion by FK866, a highly specific inhibitor of the NAD salvage pathway, can affect pattern recognition receptor-mediated responses in macrophages. NAD+-depleted mouse bone marrow-derived macrophages (BMDMs) exhibited similar levels of proinflammatory cytokine production in response to LPS or poly (I:C) stimulation compared with untreated cells. Instead, FK866 facilitated robust caspase-1 activation in BMDMs in the presence of NLRP3-activating signals such as ATP and nigericin, a potassium ionophore. However, this FK866-mediated caspase-1 activation was completely abolished in Nlrp3-deficient macrophages. FK866 plus nigericin stimulation caused an NLRP3-dependent assembly of inflammasome complex. In contrast, restoration of NAD+ level by supplementation with nicotinamide mononucleotide abrogated the FK866-mediated caspase-1 cleavage. FK866 did not induce or increase the expression levels of NLRP3 and interleukin (IL)-1β but drove mitochondrial retrograde transport into the perinuclear region. FK866-nigericin-induced mitochondrial transport is critical for caspase-1 cleavage in macrophages. Consistent with the in vitro experiments, intradermal coinjection of FK866 and ATP resulted in robust IL-1β expression and caspase-1 activation in the skin of wild-type, but not Nlrp3-deficient mice. Collectively, our data suggest that NAD+ depletion provides a non-transcriptional priming signal for NLRP3 activation via mitochondrial perinuclear clustering, and aging-associated NAD+ decline can trigger NLRP3 inflammasome activation in ATP-rich environments.

烟酰胺腺嘌呤二核苷酸（NAD+）是众多氧化还原与非氧化还原型NAD+依赖性酶促反应中的关键辅因子。细胞内NAD+水平随年龄增长持续下降，但其在髓系细胞先天免疫潜能中的作用仍不明确。本研究探讨了NAD补救合成途径（NAD salvage pathway）的高特异性抑制剂FK866介导的NAD+耗竭是否会影响巨噬细胞的模式识别受体（pattern recognition receptor）介导的免疫应答。经FK866处理发生NAD+耗竭的小鼠骨髓来源巨噬细胞（bone marrow-derived macrophages, BMDMs），在受到脂多糖（LPS）或聚肌胞苷酸（poly(I:C)）刺激时，其促炎细胞因子的产生水平与未处理对照组细胞无显著差异。与之相反，在存在ATP、尼日利亚菌素（nigericin，一种钾离子载体）这类NLRP3激活信号的条件下，FK866可显著增强BMDMs中的半胱天冬酶-1（caspase-1）活化。然而，该FK866介导的半胱天冬酶-1活化在Nlrp3缺陷型巨噬细胞中被完全抑制。FK866联合尼日利亚菌素刺激可诱导依赖于NLRP3的炎性体（inflammasome）复合物组装。反之，通过补充烟酰胺单核苷酸（nicotinamide mononucleotide）恢复细胞内NAD+水平，可抵消FK866介导的半胱天冬酶-1剪切活化。FK866既未诱导也未上调NLRP3与白细胞介素（IL）-1β的表达水平，而是促使线粒体发生逆行转运并聚集于核周区域。FK866联合尼日利亚菌素诱导的线粒体转运过程，对巨噬细胞内的半胱天冬酶-1剪切活化至关重要。与体外实验结果一致，在野生型小鼠皮肤内皮内联合注射FK866与ATP，可诱导显著的IL-1β表达与半胱天冬酶-1活化，但该效应在Nlrp3缺陷型小鼠中未出现。综上，本研究数据表明，NAD+耗竭可通过线粒体核周聚集为NLRP3活化提供非转录型激活信号，而衰老相关的NAD+水平下降可在ATP富集的微环境中触发NLRP3炎性体的活化。