Mesenchymal Stromal Cells, Autophagy, and the Host Response to Systemic Bacterial Infection

项目摘要：败血症是一种疾病过程，通过煽动引起显著的发病率和死亡率。 全身炎症反应。入侵生物(S)激活宿主的免疫细胞，以努力 根除感染。然而，这种促炎反应往往持续存在，导致器官功能障碍。 以及随后的死亡。间充质基质细胞(MSCs)和自噬过程是 对败血症的反应。自噬是受损蛋白质和细胞器周转的途径。 (例如线粒体)，它可以促进在各种疾病条件下的存活。对人类健康至关重要的基因 自噬的调节包括Beclin 1。MSCs是一群细胞，最初是从骨骼中分离出来的 骨髓，这是基于细胞治疗的一个很有前途的平台。我们已经证明，骨髓间充质干细胞可以提高患者的存活率 通过减少器官损伤，改善细菌清除，与免疫细胞相互作用和 促进炎症消退。低剂量一氧化碳暴露可诱导体内自噬，而低剂量一氧化碳暴露对 在脓毒症中，协同条件对MSCs自噬反应及其对MSC功能的影响需要 调查。我们假设自噬是调节CO(体外)能力的关键媒介。 在脓毒症实验模型中，通过MSCs的细胞间和旁分泌作用增强MSC的功能。至 检验我们的假设，我们提出了三个目标。在目标1中，我们将破译自噬的重要性 MSC体外CO预适应后，脓毒症时增强MSC功能的途径。MSCs将成为 从Beclin1/-和野生型(WT)小鼠获得的，或Beclin1将在WT MSCs中沉默(ShBeclin1)，以及 然后在盲肠结扎和穿孔(CLP)后给WT小鼠注射，这是一种实验性的脓毒症模型。我们会 确定在自噬蛋白缺陷的MSCs中，CO预适应的增强功能是否丢失。 我们还将研究一氧化碳对WT和自噬蛋白缺陷的MSCs促进细菌生长的影响 清除、消退炎症、保存器官功能和存活。在目标2中，我们将确定 细胞外小泡(EVS)在CO预适应MSCs旁分泌中的作用 研究自噬在这一反应中的重要性。EVS将从WT来源的MSCs中分离出来 Beclin1/-小鼠，或从shBeclin1 MSCs中提取，并在脓毒症发生后给予WT小鼠。在《目标3》中，我们将 确定一氧化碳对骨髓间充质干细胞体外预适应是否改善自噬蛋白的结果 败血症期间的缺陷小鼠。经CO预处理的WT MSCs将接种于Beclin1/-小鼠，并 在CLP诱导的脓毒症后，自噬被药物抑制的小鼠。此外，我们还将评估 来自MSCs的EVS暴露于CO，对自噬蛋白缺陷小鼠的脓毒症结局的影响。如果成功， 这一应用将阐明MSCs改善宿主对脓毒症反应的机制。澄清 自噬在MSCs中的重要性，以及CO对MSC功能的增强也将促进我们的 了解骨髓间充质干细胞的生物学并优化其潜在的治疗用途。

PROJECT ABSTRACT: Sepsis is a disease process that causes significant morbidity and mortality by inciting a systemic inflammatory response. The invading organism(s) activate immune cells of the host, in an effort to eradicate the infection. However, this pro-inflammatory response often persists, leading to organ dysfunction and subsequent death. Mesenchymal stromal cells (MSCs), and the autophagy process, are key mediators of the response to sepsis. Autophagy represents a pathway for the turnover of damaged proteins and organelles (such as mitochondria), which can promote survival under various disease conditions. Genes critical for the regulation of autophagy include Beclin 1. MSCs are a population of cells, originally isolated from the bone marrow, that are a promising platform for cell-based therapy. We have shown that MSCs improve survival in murine sepsis by decreasing organ injury, improving bacterial clearance, interacting with immune cells and promoting resolution of inflammation. Low-dose CO exposure induces autophagy in vivo, however the impact of CO conditioning on the autophagy response in MSCs, and its effect on MSC function during sepsis, requires investigation. We hypothesize that autophagy is a key mediator in regulating the ability of CO (ex vivo) to enhance MSC function in experimental models of sepsis, both by cell-to-cell and paracrine actions of MSCs. To test our hypothesis, we propose three aims. In Aim 1, we will decipher the importance of the autophagy pathway, for enhanced MSC function during sepsis, after CO pre-conditioning of MSCs ex vivo. MSCs will be harvested from Beclin1+/– and wild-type (WT) mice, or Beclin1 will be silenced (shBeclin1) in WT MSCs, and then administered to WT mice after cecal ligation and puncture (CLP), an experimental model of sepsis. We will determine whether the enhanced function of CO pre-conditioning is lost in autophagy protein deficient MSCs. We will also study the effect of CO on WT and autophagy protein deficient MSCs to promote bacterial clearance, resolution of inflammation, preservation of organ function, and survival. In Aim 2 we will determine the role of extracellular vesicles (EVs) in the paracrine actions of MSCs pre-conditioned with CO, and investigate the importance of autophagy in this response. EVs will be isolated from MSCs derived from WT and Beclin1+/– mice, or from shBeclin1 MSCs, and given to WT mice after the onset of sepsis. In Aim 3, we will determine whether ex vivo pre-conditioning of MSCs with CO improves the outcome of autophagy protein deficient mice during sepsis. WT MSCs pre-conditioned with CO, will be administered to Beclin1+/– mice, and mice in which autophagy is pharmacologically inhibited, after CLP-induced sepsis. In addition, we will assess EVs derived from MSCs exposed to CO, on sepsis outcome in autophagy protein deficient mice. If successful, this application will elucidate mechanisms by which MSCs improve the host response to sepsis. Elucidating the importance of autophagy in MSCs, and the enhancement of MSC function by CO will also advance our understanding of MSC biology and optimize their potential therapeutic use.