免疫性血小板减少症中DNA去甲基化在LKB1调控髓系抑制细胞中的作用及机制研究

Myeloid-derived suppressor cells (MDSC) are heterogeneous immature cells and natural inhibitors of adaptive immunity. Metabolic changes within MDSC elucidate a direct influence on immunologic consequences of their suppressive activity. Our previous study demonstrated that impaired function of MDSC participated in the pathogenesis of immune thrombocytopenia（ITP）, and that altered mitochondrial physiology was detected in patient’s MDSC. The liver kinase B1 (LKB1, encoded by Stk11) is a bioenergetic sensor that controls cell metabolism and growth. Deficiency in LKB1 leads to defective mitochondrial fitness and profoundly reduced fatty acid β-oxidation. We then found that ITP patients had significantly lower expression of LKB1 in MDSC, which could be up regulated concurrently with enhanced mitochondrial action by DNA demethylation. Epigenetic modification orchestrates proper metabolic rewriting and immune regulation. It was shown by our group and others that DNA demethylation promoted megakaryocyte maturation and platelet production; while it also entailed induction of regulatory T cells and shift of Th1-Th17/Th2 cytokine balance towards an anti-inflammatory milieu. Here, we hypothesize that intact control of MDSC metabolism by LKB1 contributes to the homeostatic regulation of immune responses, which could be potentially affected by DNA methylation status in ITP. By adopting the CRISPR/Cas9 gene editing strategy and Cre-LoxP conditional knock-out model, respectively, we will investigate both in vitro and in vivo how LKB1 modulation influences the response to DNA demethylating agents, as well as its underlying molecular mechanism.

髓系抑制细胞（MDSC）是一群具有抑制作用的固有免疫细胞。我们前期研究证实MDSC功能异常参与免疫性血小板减少症（ITP）的失耐受机制，且ITP患者MDSC存在线粒体代谢障碍。肝脏激酶B1（LKB1）是调控细胞能量代谢的重要分子，课题组进一步发现ITP患者MDSC中LKB1的转录显著低于正常人，DNA去甲基化治疗可上调其表达，并增强MDSC线粒体储备。我们据此推测LKB1对MDSC的异常调控可能参与ITP免疫失衡，而DNA去甲基化治疗可纠正这种异常，重塑MDSC抑制功能及患者免疫耐受。我们将借助ITP临床标本、细胞培养和动物实验，以DNA去甲基化对MDSC的免疫调控为切入点，分析患者MDSC中LKB1甲基化水平，通过CRISPR/Cas9靶向去甲基化基因编辑进行体外干预，并构建主动型ITP小鼠模型，全面探讨DNA去甲基化在LKB1调控MDSC中的作用及分子机制，为ITP的诊疗提供新思路。

原发免疫性血小板减少症（ITP）是临床最常见的获得性自身免疫性出血性疾病，严重威胁患者生命健康和生活质量。髓系抑制细胞（MDSCs）是一群髓系祖细胞来源的异质性未成熟细胞，其数量和功能异常参与ITP免疫失衡。MDSCs正常代谢活动的维持是其发挥免疫抑制功能的基础。本研究借助临床标本，细胞培养和ITP主动模型小鼠，以DNA去甲基化治疗对MDSCs的免疫调控为切入点，通过体外和体内实验探究了低剂量地西他滨对MDSCs的影响及其分子机制。结果表明：.1. ITP患者存在MDSCs代谢及功能异常，DNA去甲基化治疗促进MDSCs扩增并调节其功能分子表达，增强MDSCs有氧代谢水平从而改善其免疫抑制功能；.2. 肝脏激酶B1（LKB1）调控MDSCs代谢及功能稳态，ITP患者MDSCs中LKB1表达水平降低，DNA去甲基化治疗增强MDSCs中LKB1信号通路及抑制性细胞因子的表达，LKB1-shRNA转染可有效阻断DNA去甲基化治疗对MDSCs有氧代谢及免疫抑制功能的纠正作用；.3. 通过构建主动型ITP小鼠模型，在体内证明过继性转输地西他滨处理的MDSCs，能够显著改善小鼠的血小板减少症状。然而，当转染LKB1-shRNA的MDSCs移植到ITP小鼠体内时，DNA去甲基化治疗缓解血小板减少症的作用被抵消。.综上所述，我们的研究表明，MDSCs有氧代谢受损参与了ITP的发病机制，DNA去甲基化治疗对MDSCs代谢的调节作用有助于改善其免疫抑制功能。这为DNA去甲基化治疗诱导部分ITP患者持续缓解提供了可能的解释，为难治性ITP的临床诊疗提供了新思路。