The splenic CD4+ T cells mediate myocardial ischemia-reperfusion injury

 描述(由申请人提供)：越来越多的证据表明，在缺血和再灌流(I/R)期间，CD4+T细胞介导了中性粒细胞募集和器官损伤。然而，循环中或组织中的CD4+T细胞群促进心肌I/R损伤的确切机制尚不完全清楚。在肾脏和脑中，已有报道脾在介导I/R损伤中的作用。但是，在心肌再灌注损伤急性期，脾在介导炎症反应中的作用尚不清楚。以往有关脾的研究主要集中在脾尤其是脾单个核细胞在心肌梗死后重塑和I/R损伤后期(再灌流24小时)中的作用。与心肌梗死后重构不同，我们发现在缺血再灌注急性期，循环中的淋巴细胞和单核细胞减少。心肌梗死范围在再灌流后1小时内确定，而不是再灌流24小时。因此，再灌注1小时内的炎症反应在导致心肌组织坏死中更为关键。我们的初步研究表明，HMGB1和线粒体DNA(MtDNA)通过作用于RAGE和/或可能的TLR9受体，从缺血心肌中释放出来，并在再灌流时激活脾细胞。I/R前脾切除可缩小心肌梗死面积。将正常脾细胞急性过继转移到去脾小鼠的血流中，使梗塞面积恢复到对照小鼠的水平。这些结果表明，脾通过HMGB1/mtDNA−RAGE/TLR9通路在心肌I/R损伤中起中心作用。因此，我们假设，在再灌流期间，脾中的CD4+T细胞通过心-脾轴被激活，并对心肌I/R损伤起重要作用。为了验证这些假说，我们将首先确定脾CD4+T细胞在心肌I/R损伤中的作用，方法是使用切除或不切除脾的WT小鼠，并过继转移WT、RAGE-/-和CD-/-小鼠的脾细胞。在这些实验之后，我们将测试A2AR激动剂的脑梗塞保护作用是由于其对脾CD4+T细胞的A2AR的作用，并将开发一种脂质体系统将A2AR激动剂输送到脾中以抑制脾中的白细胞。其次，我们将确定心脾轴通过HMGB1/mtDNA(来自缺血心肌)-RAGE/TLR9(在脾CD4+T细胞上)途径引起I/R损伤。我们将采用一种较短的I/R，20分钟缺血和60分钟再灌流的小鼠模型，并用来自缺血40分钟小鼠的缺血心匀浆(IHH)或冠状动脉灌流液(CP)和在5分钟再灌流时获得的血浆来治疗小鼠。IHH、CP或血浆中的HMGB1和/或mtDNA可通过与RAGE或TLR9受体结合而激活脾中的CD4+T细胞，从而加重梗塞面积。最后，我们将确定治疗性阻断血液中的HMGB1或mtDNA是否足以阻断心脾轴，缩小心肌梗死范围并保护心功能。

 DESCRIPTION (provided by applicant): Accumulating evidence suggests that CD4+ T cells mediate neutrophil recruitment and organ injury during ischemia and reperfusion (I/R). However, the precise mechanisms by which CD4+ T cell populations, circulating or tissue resident CD4+ T cells, contribute to myocardial I/R injury are not fully understood. A role of the spleen in mediating I/R injury has been reported in the kidney and brain. But, the role of spleen in mediating inflammatory response during acute phase of myocardial reperfusion injury remains unknown. Previous studies related to the spleen are concentrated on role of the spleen, especially the splenic mononuclear cells, on post-MI remodeling and later phase (>24 hours of reperfusion) of I/R injury. Different from post-MI remodeling, we found that during acute phase of post-ischemic reperfusion, circulating lymphocytes and monocytes are decreased. The myocardial infarct size finalized within one hour of reperfusion versus 24 hours of reperfusion. Thus, inflammatory response within one-hour reperfusion is more critical in causing myocardial tissue necrosis. Our preliminary study has shown that substances, HMGB1 and mitochondrial DNA (mtDNA), were released from the ischemic myocardium and activated splenocytes upon reperfusion by acting on RAGE and/or possible TLR9 receptors. Splenectomy before I/R reduced myocardial infarct size. Acute adoptive transfer of normal splenocytes into the bloodstream of the splenectomized mice restored infarct size to that of control mice. These results indicate that the spleen plays a central role in myocardial I/R injury via HMGB1/mtDNA−RAGE/TLR9 pathway. We therefore hypothesize that splenic CD4+ T cells are activated via a cardio-splenic axis during reperfusion and contribute importantly to myocardial I/R injury. To test these hypotheses, we will first determine the role of splenic CD4+ T cells in myocardial I/R injury by using WT mice with or without splenectomy in combination with adoptive transfer of splenocytes from WT, RAGE-/- and CD4-/- mice. Following these experiments, we will test that infarct-sparing effect of an A2AR agonist is due to its action on A2ARs of splenic CD4+ T cells and will develop a liposome system to deliver the A2AR agonist to the spleen to inhibit the splenic leukocytes. Secondly, we will determine that a cardio-splenic axis causes I/R injury via a HMGB1/mtDNA (from ischemic myocardium) - RAGE/TLR9 (on splenic CD4+ T cells) pathway. We will employ a mouse model with shorter I/R, 20-min ischemia and 60-min reperfusion and treat mice with ischemic heart homogenates (IHH) or coronary perfusate (CP) from mice with 40-min ischemia and plasma acquired at 5-min reperfusion. The HMGB1 and/or mtDNA in IHH, CP or plasma will activate splenic CD4+ T cells by either binding to RAGEs or to TLR9 receptors and exacerbate infarct size. Finally, we will determine if the therapeutic blockade of HMGB1 or mtDNA in the blood will suffice to block the cardio-splenic axis, reduce myocardial infarct size and preserve cardiac function.