Interleukin-1ßces Human Endothelial Surface Expression and Trans-presentation of Interleukin-15 by Relieving let-7c-3p Suppression of Protein Translation (small RNA-Seq)

Expression of interleukin-15 (IL-15) on the surface of human graft endothelial cells (ECs) bound to the IL-15 receptor a (IL-15Ra) subunit can increase the activation of cytotoxic T lymphocytes (CTLs), potentiating allograft rejection. Our previous work showed that surface expression of this protein complex could be induced by alloantibody-mediated complement activation through increased IL-1b synthesis, secretion, and autocrine/paracrine IL-1-mediated activation of NF-kB. Here we report that cultured human ECs express 8 differently spliced IL-15 transcripts. Remarkably, IL-1β does not alter the expression level of any IL-15 transcript but induces surface expression independently of RNA Polymerase II-mediated transcription while requiring new protein translation. Mechanistically, IL-1β causes an NF-kB-mediated reduction in the level of microRNA Let-7c-3p, thereby relieving a block of translation of IL-15 surface protein. Let7c-3p antimir can induce EC surface expression of IL-15/IL-15Ra in the absence of complement activation or of IL-1, enabling IL-15 trans-presentation to boost CD8 T cell activation. Because of the complexity we have uncovered in IL-15 regulation, we recommend caution in interpreting increased total IL-15 mRNA or protein levels as a surrogate for trans-presentation. Cultured human endothelial colony forming cells were treated with IL1β or TNF or no treatment for 6 hours following 48 hours of IFNγ pre-treatment or no pre-treatment. Cells were lysed and RNA purified with Qiagen miRneasy.

人移植内皮细胞（graft endothelial cells, ECs）表面表达的、与白细胞介素-15受体α亚基（IL-15Rα）结合的白细胞介素-15（IL-15），可增强细胞毒性T淋巴细胞（CTLs）的活化，进而加重同种异体移植排斥反应。我们既往的研究表明，该蛋白复合物的表面表达可通过同种抗体介导的补体激活，上调IL-1β的合成与分泌，并经自分泌/旁分泌IL-1介导的核因子κB（NF-κB）活化而被诱导。本研究报道，体外培养的人内皮细胞可表达8种不同剪接异构体的IL-15转录本。值得注意的是，IL-1β并未改变任何IL-15转录本的表达水平，却可在不依赖RNA聚合酶II（RNA Polymerase II）介导的转录的情况下诱导其表面表达，且这一过程需要新的蛋白质翻译。从机制上来说，IL-1β可通过NF-κB介导的途径降低微小RNA Let-7c-3p的水平，从而解除对IL-15表面蛋白翻译的阻断。Let-7c-3p抗微小RNA寡核苷酸（antimir）可在无需补体激活或IL-1存在的情况下，诱导人内皮细胞表面IL-15/IL-15Rα的表达，进而实现IL-15跨呈递以增强CD8阳性T细胞的活化。鉴于我们在IL-15调控中发现的复杂性，我们建议在将总IL-15 mRNA或蛋白水平的升高作为IL-15跨呈递的替代标志物进行解读时需谨慎。我们对体外培养的人内皮集落形成细胞进行了如下处理：在经48小时干扰素γ（IFNγ）预处理或未预处理后，分别用IL-1β、肿瘤坏死因子（TNF）处理或不予处理，持续6小时。随后收集细胞并裂解，使用凯杰（Qiagen）miRNeasy试剂盒纯化RNA。