HDAC2通过p21调控中性粒细胞NETs形成及其在机体抗感染免疫调控中的作用机制研究

Infection is still a major problem that afflicts human health. Neutrophil Extracellular Trap (NET) formation is an important mechanism for organisms to capture and clear off pathogens, but how it is regulated remains unclear. NET is a network of chromatin DNA and proteins with antibacterial activities released by neutrophils usually when stimulated by infections, and its formation relies on CDK6 activation. Our preliminary studies have demonstrated that HDAC2 deficient mice showed significantly impaired ability to clear off bacteria in blood, reduced NET formation, and increased expression of p21 that inhibits CDK6 kinase. Since it has been reported that HDAC2 suppresses P21 expression in tumor cells by forming a transcriptional repression complex with P53 that binds to the promoter region of P21 and down-regulates histone acetylation, we speculated that HDAC2 participates in immune response against infection by regulating NETosis of neutrophils via the P21-CDK6 signaling pathway. To confirm this hypothesis, this study intends to use HDAC2 knockout mouse and its primary neutrophils as models to investigate the effect of HDAC2 on neutrophil NET formation and immune response to infections by RNA interference, inhibitors, CHIP assay, reporter gene assay, etc. This study is expected to provide new theoretical basis for novel anti-infection therapies.

感染仍是困扰人类健康的重大问题，NETs是机体捕获和清除病原菌的重要机制，但其调控机制仍完全不清楚。NETs是中性粒细胞受感染因素诱导而释放出包括DNA和各种抗菌蛋白组成的一种网状结构，其形成依赖CDK6的活化。前期预实验发现，HDAC2敲除小鼠细菌清除能力显著下降，中性粒细胞NETs形成受抑，CDK6激酶抑制物p21的表达显著上调。同时，鉴于HDAC2可与p53在瘤细胞形成转录抑制复合物，并结合于p21启动子区，下调组蛋白的乙酰化水平，抑制p21的表达。我们由此提出“HDAC2通过p21-CDK6信号轴调控中性粒细胞NETs形成，参与机体抗感染免疫调控的机制”假设。为证实此假设，本研究拟以HDAC2敲除的中性粒细胞和小鼠为模型，并通过干扰、抑制剂、CHIP技术和报告基因实验等，探索HDAC2对中性粒细胞NETs形成及机体抗感染免疫调控的作用机制，为抗感染治疗提供新的理论依据。

感染是危害人类健康的重大感染性疾病的因素，严重威胁人类健康。中性粒细胞是机体抵御病原体入侵的第一道防线，在抗感染过程中发挥主力军的作用，NETs是中性粒细胞捕获和清除病原菌的重要机制，但其调控机制仍完全不清楚。我们研究发现HDAC2对中性粒细胞的吞噬功能无显著影响，但是却显著抑制中性粒细胞形成NETs的功能，显著降低清除细菌的能力。研究结果显示，干扰HDAC2和p53基因表达水平，显示p21表达显著增强，中性粒细胞形成NETs能力下降。同时，p21蛋白特异性抑制活性片段（p21 inhibitory peptide, p21inh)具有p21的活性功能，p21inh显著抑制中性粒细胞NETs形成。进一步研究发现，HDAC2 KO中性粒细胞CDK6活性下降，其下游信号活化后相关的细胞周期蛋白及P-pRb水平下降，NETs形成能力减弱。在动物研究中，HDAC2 KO小鼠显示出清除细菌能力下降以及器官载菌量上升，抗菌能力下降。本项目通过体内外实验探索出了HDAC2-p21-CDK6信号轴对中性粒细胞NETs形成及机体抗感染免疫调控的作用机制，为抗感染治疗提供新的理论依据。