调节性B细胞在心肌缺血再灌注损伤中调控炎症反应动态平衡的作用及机制研究

The maintenance of dynamic equilibrium between proinflammation and anti-inflammation is a key strategy to mitigate myocardial ischemia reperfusion injury (MIRI). Regulatory B cells (Bregs) can suppress the proinflammatory cells as well as enhance the recruitment or polarization of other immunoregulatory cells. It is essential for inflammation regulation of disease initiation and subsequent inflammation resolution. However, the involvement of Bregs in MIRI is poorly defined. We found that Bregs infiltrated the myocardium early after MIRI. B cell -deficient mice presented with less Bregs, but with more sever inflammation and larger infarction area after MIRI. Drug-induced increasement of Bregs can mitigate MIRI. Based on these evidences, we believed that Bregs can attenuate MIRI by regulating the dynamic equilibrium between proinflammation and anti-inflammation. In the present project, we plan to fully investigate the dynamic changes in amount of Bregs following MIRI, characterize the effect of Breg on the immune components and infarction area at different time points after MIRI by changing the amounts of Bregs, screen the potential cytokines mediating the immunoregulatory function of Bregs in MIRI by Milliplex assays, explore the molecular mechanism by which Bregs regulate the dynamic equilibrium of inflammation after MIRI using the candidate cytokine knockout mice or antibody blocking, and thus provide a new strategy for the therapeutic immunoregulation of MIRI.

调节促炎-抑炎反应动态平衡是减轻心肌缺血再灌注损伤(MIRI)的关键。调节性B细胞(Bregs)兼具抑制炎性细胞与促进其他免疫调节细胞募集或极化的功能，在疾病早期免疫调控及后续促炎-抑炎转化中扮演重要角色。但Bregs对MIRI的作用及机制尚不清楚。我们发现MIRI早期心脏局部Bregs水平增加。B细胞缺陷小鼠MIRI后，心脏Bregs浸润减少，局部炎性细胞及炎症因子水平增加，心肌损伤加重。诱导Bregs水平增加的药物可减轻MIRI。据此，我们认为Bregs可能通过重塑心脏局部微环境炎症动态平衡影响MIRI。为此，本项目拟系统分析MIRI后Bregs水平变化情况；改变Bregs水平对再灌注后不同时期其他免疫成分及组织损伤的影响；应用多因子检测技术筛选可能的效应分子，联合模式动物或抗体阻断效应分子的方式，探讨Bregs调节MIRI炎症动态平衡的分子机制，为MIRI的免疫调节治疗提供新策略。

再灌注损伤所致心肌死亡占全部细胞心肌的50%。无菌性炎症反应是心肌缺血再灌注损伤（MIRI）的机制之一。近年来研究显示，B细胞展现了表型的多样性，一些B细胞亚群被证明具有免疫调节作用，有减轻组织损伤的作用。但目前鲜有研究关注B细胞在MIRI中的作用。本研究旨在阐明B细胞在MIRI中的作用。首先，我们发现在心脏中，B细胞及IL10+B细胞在MIRI后明显升高。在心肌缺血再灌注后，选择性清除B细胞或B细胞缺陷小鼠心脏组织损伤及心脏病理性重构明显增加，这表现在与对照组相比，选择性清除B细胞或B细胞缺陷小鼠组织损伤积分增加，心肌细胞凋亡水平高增加，心功能恶化，心肌梗死面积增加。转录组学及组织学实验表明B细胞清除或缺乏加重了MIRI后的心脏局部炎症反应。回复实验表明B细胞的免疫调控及心脏保护作用并不完全依赖于IL10，这表现在过继输注IL10-/-B细胞到B细胞缺陷（μM）小鼠仍可以部分改善心肌损伤。机制上，我们发现B细胞可以通过细胞间相互接触的方式诱导中性粒细胞凋亡，使其被巨噬细胞吞噬，进而促进单核巨噬细胞从促炎型向修复型转变。利用小鼠缺血心肌的单细胞转录组数据进行配体-受体推断分析显示Fcer2a和CD22可能介导了B细胞与中性细胞相互作用；体外实验表明，阻断B细胞表面的Fcer2a而非CD22抑制了对中性粒细胞的凋亡诱导作用。在心肌缺血再灌注后，给予Anti-Fcer2a可以明显减少B细胞与中性粒细胞在物理空间上的接触。总体而言，本研究揭示了B细胞通过Fcer2a与中性粒细胞作用，间接影响巨噬细胞表型转化，最终发挥其在心肌缺血再灌注后的心肌保护作用。