Modulation neuroinflammation through interference of cooperative microRNA-RNA-binding protein interactions

Th 17细胞及其有效的炎性细胞因子（IL-17 A和GM-CSF）在多种免疫反应中的重要性， 硬化症（MS）和其他自身免疫性疾病的发病率。在动物MS模型中，募集Th 17细胞， 并通过β 2整合素LFA-1依赖性粘附和跨内皮迁移定位于CNS。 虽然IL-17 A和GM-CSF基因的转录调控已经得到很好的表征，但mRNA表达水平仍然存在差异。 编码这些细胞因子的细胞因子是高度不稳定的，并且必须动态调节以允许显著的基因表达。 表情我们已经证明，通过α 2整合素接合的T细胞粘附导致显著的免疫应答。 通过调节和核-胞质易位稳定编码TNF-α和IFN-β的mRNA RNA结合蛋白（RBP）HuR。我们的初步数据支持一个同样显着的扩展， IL-17 A和GM-CSF转录物半衰期通过LFA刺激的HuR依赖性机制。当 试图表征IL-17 A 3 '-HuR上潜在的竞争性microRNA（miRNA）- HuR相互作用， 在非翻译区（3 '-UTR）中，我们意外地检测到一个合作的，相互依赖的RNA稳定蛋白。 miR-466 l-3 p与HuR之间的相互作用。我们将miR-466 l-3 p靶位点定位在IL-17 A 3 '-UTR内。 阻止这种相互作用的寡核苷酸（靶位点阻断剂[TSB]）抑制LFA-1诱导的HuR-1， 依赖性IL-17 A mRNA稳定，并以精氨酸特异性方式增强IL-17 A产生。我们 由于GM-CSF mRNA的3 '-UTR含有高度保守的富含AU的元件，因此， 其包括4个潜在的miR-466 l-3 p靶位点。我们以前公布的和新的数据，以及 IL-17 A和GM-CSF在神经炎症中的致病重要性，导致我们的假设， 白细胞整联蛋白参与通过增强Th 17细胞IL-17 A和GM-CSF表达促进Th 17细胞IL-17 A和GM-CSF表达 HuR和miR-466 l-3 p与它们的3 '-UTR的协同结合，并且这种有效的促炎性作用可能与它们的3'-UTR结合有关。 开关适合于新治疗靶向。目前的具体建议是：（1）定位miR-466 l-3 p 在GM-CSF 3 '-UTR的靶位点，并产生有效的，特异性TSB，使用互补的分子 方法包括（a）MS 2-TRAP 3 '-UTR/miRNA下拉，和（B）pBB B β珠蛋白RNA报告基因稳定性 （2）确定选择性阻断miR-466 l-3 p与IL-17 A和GM-11相互作用的影响。 CSF转录物对慢性髓鞘少突胶质细胞糖蛋白（MOG）特异性2D 2免疫病理学的影响 转基因EAE模型，和复发、缓解、蛋白脂质蛋白肽（PLP）免疫的EAE模型， 评价EAE临床评分以及CNS IL-17 A和GM-CSF mRNA和蛋白水平。的新奇 这种新描述的合作miRNA-RBP相互作用，以及我们测试针对这种作用的抑制剂的能力， 在神经炎症疾病模型中的协同性，使其成为分子和高度转化的 探索性R21项目。我们希望这项工作能指导更广泛的转录后调控工作， 病原性细胞因子表达，并定义了一种新的治疗靶向机会。

The importance of Th17 cells, and their potent inflammatory cytokines (IL-17A and GM-CSF), in multiple sclerosis (MS) and other autoimmune diseases is established. In animal MS models, Th17 cells are recruited and localized to the CNS through 2 integrin LFA-1-dependent adhesion and transendothelial migration. Although transcriptional regulation of the IL-17A and GM-CSF genes has been well characterized, the mRNAs encoding these cytokines are highly labile and must be dynamically regulated to allow significant gene expression. We have demonstrated that T cell adhesion through 2 integrin engagement results in marked stabilization of mRNAs encoding TNF- and IFN-, through modulation and nuclear-to-cytosolic translocation of the RNA-binding protein (RBP) HuR. Our preliminary data support an equally remarkable extension of the IL-17A and GM-CSF transcript half-lives through an LFA-stimulated, HuR-dependent mechanism. When attempting to characterize potential competitive microRNA (miRNA)- HuR interactions on the IL-17A 3'- untranslated region (3'-UTR), we unexpectedly detected a cooperative, interdependent RNA- stabilizing interaction between miR-466l-3p and HuR. We mapped the miR-466l-3p target site within the IL-17A 3'-UTR. An oligonucleotide preventing this interaction (target site blocker [TSB]) inhibits LFA-1-induced, HuR- dependent IL-17A mRNA stabilization, and enhanced IL-17A production, in a cytokine-specific manner. We intend to define the same for GM-CSF, as its mRNA's 3'-UTR contains a highly conserved AU-rich element which includes 4 potential miR-466l-3p target sites. Our previously published and new data, and the pathogenic importance of IL-17A and GM-CSF in neuroinflammation, have led to our hypothesis, that leukocyte integrin engagement promotes Th17 cell IL-17A and GM-CSF expression via enhanced cooperative binding of HuR and miR-466l-3p to their 3'-UTRs, and that this potent pro-inflammatory switch is amenable to novel therapeutic targeting. Specific proposals now are to: (1) map the miR-466l-3p target site in the GM-CSF 3'-UTR, and generate an effective, specific TSB, using complementary molecular approaches including (a) MS2-TRAP 3'-UTR/miRNA pulldowns, and (b) pBBB  globin RNA reporter stability assays; and (2) determine the impact of selectively blocking miR-466l-3p's interaction with the IL-17A and GM- CSF transcripts on immunopathology in a chronic, myelin oligodendrocyte glycoprotein (MOG)-specific 2D2 transgenic EAE model, and a relapsing, remitting, proteolipid protein peptide (PLP)-immunized EAE model, evaluating EAE clinical scores, as well as CNS IL-17A and GM-CSF mRNA and protein levels. The novelty of this newly described cooperative miRNA-RBP interaction, and our ability to test inhibitors directed at this cooperativity in neuroinflammation disease models, makes this both a molecular and a highly translational exploratory R21 project. We hope this work directs more extensive efforts in posttranscriptional regulation of pathogenic cytokine expression, and defines a novel therapeutic targeting opportunity.