Role of Regulatory T-Lymphocytes in Chronic Heart Failure

 描述(由申请人提供)： 目前还没有大规模的免疫调节性临床治疗心力衰竭(HF)。到目前为止，治疗性免疫调节的主要焦点一直是中和炎症介质，如肿瘤坏死因子-α，这一方法不幸在临床试验中失败。最近，我们描述了全球免疫细胞网络的深刻重构和迄今未被认识的慢性缺血性心力衰竭的不利心脾轴，它促进了衰竭心脏中免疫细胞介导的组织损伤。这一新的发现表明，直接针对在HF中改变的特定白细胞群可能是治疗免疫调节的更好方法。调节性T细胞(Tregs)是一种CD4T淋巴细胞，表达Foxp3转录因子，以一种接触性、非抗原特异性的方式抑制免疫细胞反应；它们在慢性心力衰竭中的病理生理作用尚不清楚。在我们的初步研究中，我们出人意料地发现，在HF中，Treg强大地渗透到衰竭的心脏，而不是抗炎从而保护心脏，相反，它反常地表现出促炎特征和显著的疾病介质功能，赋予了大量的抗血管生成、促纤维化和促炎作用。基于这些数据，我们假设功能障碍的Treg在心力衰竭的左心室(LV)重构、毛细血管疏松、炎症和疾病进展中是必不可少的，从而 是免疫调节的关键细胞靶点。三个目标将检验这一假设。在目标1中，我们将描述在冠脉结扎和心力衰竭的C57BL/6小鼠和假手术对照组中Treg的全球运输和Treg促炎信号的变化。Tregs等T细胞亚群，在结扎后的不同时间点，通过流式细胞术和免疫组织化学方法检测心、血、脾和纵隔淋巴结的炎性变化，并与LV和脾的重塑和炎症相关。BrdU标记可作为Treg体内增殖的指标。此外，利用人类心力衰竭生物样本库，我们将确定Tregs在人类心力衰竭心脏中的丰度和分布，并与匹配的非心力衰竭对照组相比，测定患有心力衰竭(射血分数降低)的门诊患者的循环Tregs(和其他T细胞)的情况。在目标2中，我们将通过选择性(和可逆地)去除慢性心力衰竭Foxp3-DTR TG小鼠的Tregs，然后评估其对免疫细胞谱、炎症、循环和骨髓内皮祖细胞、体内血管生成和LV的影响，来确定Tregs在慢性心衰小鼠左室重构和新血管形成中的作用。 改建。在平行研究中，将使用体外试管形成试验比较来自假手术小鼠、心力衰竭小鼠和心力衰竭小鼠的脾树突在消融和随后重建树突细胞时的相对抗血管生成作用。在目标3中，我们将确定功能障碍的Treg是否是慢性心力衰竭心脾轴受损的重要介质。将Foxp3-DTR TG小鼠早期或晚期心衰(对应于Treg功能障碍的不同阶段)的CD45.2单核脾细胞过继转移到CD45.1幼稚小鼠体内，移植前进行Treg消融或不进行Treg消融。然后，将在受体小鼠中测量细胞转移对LV/脾重构、炎症和T细胞/免疫细胞图谱的长期影响。同时，我们将评估来自HF和Sham小鼠的脾Tregs的能力 通过使用TNFR1-/-和TNFR2-/-HF小鼠的脾T细胞进行类似的研究，在体外抑制CD4和CD8脾T淋巴细胞的激活，以及TNFR信号在HF Treg功能障碍中的作用。通过明确Tregs在病理性心脏重塑中的作用，这些研究将进一步加深我们对慢性缺血性心力衰竭炎症激活的细胞基础的理解，并为最近发现的促炎和组织损伤的心脾轴的基本基础提供创新的视角。此外，通过提供直接 有证据表明Tregs在心力衰竭中是抗血管生成和(矛盾的)促炎介质，这些研究将确定针对细胞类型而不是细胞因子特定的免疫调节的新靶点。

 DESCRIPTION (provided by applicant): No large-scale immunomodulatory clinical therapies currently exist for heart failure (HF). To date, the primary focus of therapeutic immunomodulation has been to neutralize inflammatory mediators such as tumor necrosis factor-α, an approach that unfortunately failed in clinical trial. Recently, we described profound remodeling of the global immune cell network and a heretofore-unappreciated adverse cardiosplenic axis in chronic ischemic HF that promotes immune cell-mediated tissue injury in the failing heart. This novel discovery suggests that directly targeting specific leukocyte populations that are altered in HF may represent a better approach to therapeutic immunomodulation. Regulatory T-cells (Tregs) are CD4+ T-lymphocytes that express the forkhead box protein-3 (Foxp3) transcription factor and suppress immune cell responses in a contactdependent, antigen nonspecific manner; their pathophysiological role in chronic HF is unknown. In our preliminary studies, we have unexpectedly discovered that in HF, Tregs robustly infiltrate the failing heart, and rather than being anti-inflammatory and thereby cardioprotective, paradoxically exhibit pro-inflammatory features and function prominently as disease mediators, imparting substantial anti-angiogenic, pro-fibrotic, and pro-inflammatory effects. Based on these data, we hypothesize that dysfunctional Tregs are essential for left ventricular (LV) remodeling, capillary rarefaction, inflammation, and disease progression in HF, and thereby are key cellular targets for immunomodulation. Three Aims will test this hypothesis. In Aim 1, we will delineate global Treg trafficking and alterations in Treg pro-inflammatory signaling in C57BL/6 mice with coronary ligation and HF, and in sham-operated controls. Tregs, and other T-cell subsets, and their inflammatory profiles will be defined in heart, blood, spleen, and mediastinal lymph nodes by flow cytometry and immunohistochemistry at serial time points after ligation, and correlated with LV and splenic remodeling and inflammation. BrdU labeling will index Treg proliferation in vivo. Moreover, using human HF biosample repositories, we will determine the abundance and distribution of Tregs in human failing hearts, and profile circulating Tregs (and other T-cells) in ambulatory patients with HF (with reduced ejection fraction) versus matched non-failing controls. In Aim 2, we will establish the role of Tregs in LV remodeling and neovascularization by selectively (and reversibly) depleting Tregs in Foxp3-DTR Tg mice with chronic HF, and then evaluating the effects on immune cell profiles, inflammation, circulating and bone marrow endothelial progenitor cells, in vivo angiogenesis, and LV remodeling. In parallel studies, the relative anti-angiogenic effects of splenic Tregs from sham mice, HF mice, and HF mice with ablation and subsequent reconstitution of Tregs will be compared using in vitro tube formation assays. In Aim 3, we will define whether dysfunctional Tregs are important mediators of the injurious cardiosplenic axis in chronic HF. CD45.2 mononuclear splenocytes from Foxp3-DTR Tg mice with either early or late HF, (corresponding to different stages of Treg dysfunction) will be adoptively transferred into CD45.1 naïve mice, with or without Treg ablation prior to transfer. The long-term effects of cell transfer on LV/splenicremodeling, inflammation, and T-cell/immune cell profiles will then be measured in recipient mice. In parallel, we will evaluate the ability of splenic Tregs from HF and sham mice to suppress activation of CD4+ and CD8+ splenic T-lymphocytes in vitro, and the role of TNFR signaling in HF Treg dysfunction via similar studies using splenic T-cells from TNFR1-/- and TNFR2-/- HF mice. By conclusively defining the role of Tregs in pathological cardiac remodeling, these studies will further our understanding of the cellular basis for inflammatory activation in chronic ischemic HF, and provide innovative perspectives as to the fundamental underpinnings of the recently discovered pro-inflammatory and tissue-injurious cardiosplenic axis. Moreover, by providing direct evidence for Tregs as anti-angiogenic and (paradoxically) pro-inflammatory mediators in HF, these studies will identify new targets for cell type-specific, rather than cytokine-specific, immunomodulation.