Mitochondrial transcription factor A, an endogenous danger signal, promotes TNFα release via RAGE- and TLR9-responsive plasmacytoid dendritic cells.
Mitochondrial transcription factor A, an endogenous danger signal, promotes TNFα release via RAGE- and TLR9-responsive plasmacytoid dendritic cells.
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DOI：
10.1371/journal.pone.0072354
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Crouser ED
中科院分区：
文献类型：
--
作者：
Julian MW;Shao G;Vangundy ZC;Papenfuss TL;Crouser ED
线粒体转录因子A （TFAM）从受损细胞中释放出来后，通常与线粒体DNA （mtDNA）结合并保持相关。我们假设，与mtDNA（或等效的cpg富集DNA）结合的TFAM通过参与RAGE，放大表达tlr9的浆细胞样树突状细胞（pDCs）的TNFα释放。用重组人TFAM单独或与富含cpg的DNA联合处理C57BL/6小鼠Flt3配体扩增的脾细胞，随后用ELISA检测TNFα的释放。RAGE的作用是通过可溶性RAGE或肝素预处理或使用匹配的RAGE（-/-）脾细胞来确定的。TLR9信号通过特异性TLR9阻断寡核苷酸和抑制内体加工、PI3K和NF-κB来评估。另外的研究检查了硫酸肝素部分或内皮素转换酶-1 （ECE-1）依赖的内体受体循环是否需要TFAM和CpG DNA识别。TFAM增加了脾细胞对CpGA DNA的释放，这强烈依赖于pDCs并受RAGE和TLR9受体的调节。TLR9信号通路，包括内体酸化和通过PI3K和NF-κB的信号通路，是脾细胞释放TNFα响应TFAM+CpGA DNA所必需的。有趣的是，TNFα的释放依赖于内皮素转换酶(ECE)-1，该酶可切割并激活内体中的TLR9。TFAM- cpga DNA复合物的识别依赖于硫酸肝素部分，重组TFAM Box 1和Box 2蛋白在增加TNFα释放方面是等效的。在脾细胞培养模型中，TFAM促进了TNFα的释放，这代表了体内复杂的细胞-细胞相互作用，pDCs起着关键作用。据我们所知，这项研究首次证实了依赖ece -1的TLR9内体切割是导致TNFα释放的信号通路中的关键步骤。这些发现，以及本文报道的其他发现，极大地促进了我们对线粒体危险信号触发的无菌免疫反应的理解。
Mitochondrial transcription factor A (TFAM) is normally bound to and remains associated with mitochondrial DNA (mtDNA) when released from damaged cells. We hypothesized that TFAM, bound to mtDNA (or equivalent CpG-enriched DNA), amplifies TNFα release from TLR9-expressing plasmacytoid dendritic cells (pDCs) by engaging RAGE. Murine Flt3 ligand-expanded splenocytes obtained from C57BL/6 mice were treated with recombinant human TFAM, alone or in combination with CpG-enriched DNA with subsequent TNFα release measured by ELISA. The role of RAGE was determined by pre-treatment with soluble RAGE or heparin or by employing matching RAGE (-/-) splenocytes. TLR9 signaling was evaluated using a specific TLR9-blocking oligonucleotide and by inhibiting endosomal processing, PI3K and NF-κB. Additional studies examined whether heparin sulfate moieties or endothelin converting enzyme-1 (ECE-1)-dependent recycling of endosomal receptors were required for TFAM and CpG DNA recognition. TFAM augmented splenocyte TNFα release in response to CpGA DNA, which was strongly dependent upon pDCs and regulated by RAGE and TLR9 receptors. Putative TLR9 signaling pathways, including endosomal acidification and signaling through PI3K and NF-κB, were essential for splenocyte TNFα release in response to TFAM+CpGA DNA. Interestingly, TNFα release depended upon endothelin converting enzyme (ECE)-1, which cleaves and presumably activates TLR9 within endosomes. Recognition of the TFAM-CpGA DNA complex was dependent upon heparin sulfate moieties, and recombinant TFAM Box 1 and Box 2 proteins were equivalent in terms of augmenting TNFα release. TFAM promoted TNFα release in a splenocyte culture model representing complex cell-cell interactions in vivo with pDCs playing a critical role. To our knowledge, this study is the first to incriminate ECE-1-dependent endosomal cleavage of TLR9 as a critical step in the signaling pathway leading to TNFα release. These findings, and others reported herein, significantly advance our understanding of sterile immune responses triggered by mitochondrial danger signals.
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影响因子：
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