TNFR1 Expressing Exosomes are Critical Mediators of Pathological Immune Activation in the Spleen post-Myocardial Infarction

项目摘要 免疫激活是心肌梗死（MI）的标志，并决定梗死愈合和左心室（LV） 重塑在临床前和临床研究中已经确定，在梗死后立即免疫 来自脾和骨髓（BM）的细胞进入体循环并运输到缺血的 心中这表明来自受损心脏的损伤信号被传递到淋巴系统， 造血组织启动心脏特异性免疫反应。然而， 这些信号被转移到免疫丰富的小生境，如脾脏，是未知的。 最近的研究表明，外泌体，30-100纳米大小的膜囊泡， 细胞间通讯载体，可以穿梭mRNA，miRNA和蛋白质到远处的组织， 生理和病理免疫激活。还已知循环外泌体水平增加 在肌细胞损伤期间，并且在其货物中含有肌节蛋白、细胞溶质蛋白和线粒体蛋白。尽管 这种理解，不知道外来体是否也作为抗原载体携带损伤- 相关的蛋白质信号从缺血心肌到脾脏，用于随后的免疫激活。 事实上，我们的初步结果清楚地表明，静脉内施用由细胞释放的外泌体， 缺血心脏（与假手术相比）诱导i）收缩功能障碍（增加的终末- 舒张期和收缩末期容积以及射血分数降低），ii）与损伤相关的基因表达 信号（S100 A8，S100 A9和嗜酸性粒细胞趋化因子），iii）脾重塑，和iv）先天性巨噬细胞浸润 和适应性免疫细胞。MI外泌体表达肿瘤坏死因子 受体-1（TNFR 1，而不是TNFR 2），一种经典的促炎信号分子， 临床上已显示与HF严重程度和心功能不全相关。重要的是，静脉注射 注射从1d MI TNFR 1-/-小鼠中分离的外泌体未能诱导幼稚小鼠的心功能障碍 这表明外来体TNFR 1在介导MI后免疫激活中的有效且先前未知的作用 和心脏功能障碍。因此，这使我们假设外泌体携带心脏抗原来介导 MI期间脾免疫细胞活化对于免疫细胞介导的病理性LV重塑至关重要， 这些作用依赖于外来体TNFR 1的表达。重要的是，这些是关键的细胞靶点 免疫调节我们将通过i）确定脾脏的病理生理作用来检验这一假设 在MI后介导外泌体加工中，ii）定义外泌体中囊泡TNFR 1的来源和作用， iii）确定外来体TNFR 1在介导免疫原性心功能障碍中的作用， 发信号。

Project Summary Immune activation is a hallmark of myocardial infarction (MI) and dictate infarct healing and left ventricular (LV) remodeling. It is established, both in preclinical and clinical studies, that immediately after infarction immune cells from the spleen and the bone-marrow (BM) egress into the systemic circulation and traffic to the ischemic hearts. This suggests that the damage signals from the injured hearts are communicated to the lymphoid and the hematopoietic tissues to initiate cardiac-specific immune responses. However, the mechanisms by which these signals are transferred to the immune-rich niches such as the spleen are not known. Recent studies have shown that exosomes, membranous vesicles of 30-100 nm size, are potent intercellular communication vehicles that can shuttle mRNA, miRNA, and proteins to the distant tissues for physiological and pathological immune activation. It is also known that circulating exosomal levels increase during myocyte damage and contain sarcomeric, cytosolic and mitochondrial proteins in their cargo. Despite this understanding, it is not known if exosomes also serve as the antigenic vehicles to carry damage- associated protein signals from the ischemic myocardium to the spleen for subsequent immune activation. Indeed, our preliminary results clearly show that intravenous administration of exosomes released by the ischemic hearts (as compared to sham) into the naïve mice induce i) systolic dysfunction (increased end- diastolic and end-systolic volumes and decreased ejection fraction), ii) gene expression of damage associated signals (S100A8, S100A9 and eotaxin) in the left-ventricles, iii) splenic remodeling, and iv) infiltration of innate and adaptive immune cells in the myocardium. Moreover, MI exosomes expressed tumor necrosis factor receptor-1 (TNFR1; and not TNFR2) on their membranes, a classical pro-inflammatory signaling molecule that have been shown to correlate with HF severity and cardiac dysfunction clinically. Importantly, intravenous injection of exosomes isolated from 1d MI TNFR1-/- mice failed to induce cardiac dysfunction in naïve mice suggesting a potent, and previously unknown, role of exosomal TNFR1 in mediating immune activation post-MI and cardiac dysfunction. Therefore, this led us to hypothesize that exosomes carry cardiac antigens to mediate splenic immune cell activation during MI, are critical for immune cell mediated pathological LV remodeling, and these effects are dependent upon the exosomal TNFR1 expression. Importantly, these are key cellular targets for immunomodulation. We will test this hypothesis by i) determining the pathophysiological role of the spleen in mediating exosomal processing post-MI, ii) defining the source and role of vesicular TNFR1 in exosome mediated cardiac dysfunction, and iii) determining the role of exosomal TNFR1 in mediating immunogenic signaling.