免疫性血小板减少症中糖皮质激素受体调控MDSC代谢及功能的研究

Primary immune thrombocytopenia (ITP) is the most common autoimmune bleeding disorder characterized by increased platelet destruction due to severe loss of immune tolerance. Current studies on ITP reveal accumulated focus on impaired immune modulatory cells such as Tregs and Bregs, etc. As a negative regulator bridging native and adaptive immunity, myeloid-derived suppressor cells (MDSCs) might also participate in this thrombocytopenic syndrome. Our previous work demonstrated that dysfunction of MDSCs played a role in the pathogenesis of ITP, and that HD-DXM raised MDSC number and corrected its suppressive function in the management of ITP. Nevertheless, the underlying therapeutic mechanism of HD-DXM is not fully understood. Glucocorticoid receptors (GR) are nuclear transcription factors located in the cytosol. Recent study has established that, in eukaryotic cells, translocation of GR occurs from the cytosol to the mitochondria in response to corticosteroids, which elicit a direct influence on mtDNA transcription and mitochondrial physiology. Here, we hypothesize that GR regulates the expression of mtDNA and therefore oxidative metabolism of MDSCs in patients with ITP. By digging into the samples of ITP patients and constructing a conditional KO mouse model, this study will explore the influence of GR on MDSC function, discuss the immune pathogenesis of ITP from a metabolic perspective and shed new light on the molecular mechanism of glucocorticoid action.

免疫失耐受是原发免疫性血小板减少症（ITP）中血小板破坏的重要机制。免疫调节细胞功能异常逐渐成为ITP免疫失衡的研究热点。髓源抑制性细胞（MDSCs）是一群具有负调控作用的固有免疫细胞，参与多种自身免疫性疾病的发生。申请人前期研究发现ITP患者外周MDSCs较正常人数量减少且存在功能缺陷，大剂量地塞米松可纠正ITP患者MDSCs的不足并增强其免疫抑制功能，但分子机制尚不明确。糖皮质激素受体（GR）是位于靶细胞胞浆中的核转录因子，真核细胞的GR可向线粒体转移并在糖皮质激素的作用下，影响线粒体DNA的转录和氧化磷酸化。本研究首次提出GR通过调控MDSCs线粒体DNA的表达，增强细胞有氧代谢及生物学功能这一假说，拟以GR为切入点，研究ITP患者MDSC代谢特征并构建Cre-LoxP条件性敲除小鼠，从体外和体内两方面探讨干预GR对MDSCs功能的影响及其分子机制，为ITP的诊疗提供新思路。

免疫性血小板减少症（ITP）是一种自身免疫性出血性疾病，病理表现包括抗血小板抗体诱导血小板过度破坏和产生不足；同时，T细胞亚群失衡和功能受损也参与了ITP的发病机制，如细胞毒性T细胞的积累、调节性T细胞的减少等。髓源抑制性细胞（MDSCs）是起源于髓系祖细胞的未成熟前体，其作用是抑制T细胞增殖、激活和应答以及调节性T细胞的招募和扩展。MDSCs的代谢变化与其在免疫反应中的抑制作用息息相关。我们的研究表明：1.大剂量地塞米松（HD-DXM）可纠正ITP患者MDSCs功能受损，而激素治疗无效者MDSCs功能未能恢复。此外，HD-DXM治疗无效患者的MDSCs中线粒体活性下降、线粒体基因转录减少，伴有脂肪酸氧化（FAO）关键酶肉毒碱棕榈酰转移酶-1（CPT-1）水平下降。2.通过CPT-1抑制剂etomoxir阻断FAO会抑制HD-DXM治疗有效患者的MDSCs的免疫抑制功能。用etomoxir干预ITP患者及正常对照诱导后的MDSCs，发现在缓解组中，经etomoxir处理的MDSCs对CD4+ T细胞分裂的阻断能力显著降低；而在治疗无效组中并无此现象。CHIP-qPCR结果显示治疗无效组的ITP患者MDSCs中糖皮质激素受体（GR）与mtDNA D-loop区域的相互作用明显减少，线粒体mtDNA转录受损。3.在被动型ITP小鼠模型及主动型ITP小鼠模型中，证明了输注敲除GR的MDSCs的小鼠血小板计数及调节性T细胞的比例明显低于小干扰RNA阴性对照组，同时，我们发现敲除组的ND-1、ND-2、ND-3、ND-6、Cyt-b、COX-1、ATP-6和CPT-1的表达水平均显著降低，表明在DEX作用的MDSCs中，GR下调也会影响mt-DNA和CPT-1的表达。综上所述，我们的研究表明MDSCs有氧代谢受损参与了ITP糖皮质激素抵抗的发病机制，糖皮质激素受体通过影响线粒体DNA转录和有氧代谢以调控MDSCs的免疫抑制功能。