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

项目摘要：脓毒症是一种疾病过程，通过刺激 全身炎症反应入侵的生物体激活宿主的免疫细胞， 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（MSC）和自噬过程是细胞增殖的关键介质。 对败血症的反应自噬代表了受损蛋白质和细胞器周转的途径 (such作为线粒体），其可以在各种疾病条件下促进存活。关键基因 调节自噬的包括Beclin 1。骨髓间充质干细胞是一群细胞，最初从骨骼中分离出来， 骨髓，这是一个有前途的平台，为基于细胞的治疗。我们已经证明，MSC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， 促进炎症消退。低剂量CO暴露在体内诱导自噬，然而， 一氧化碳调节骨髓间充质干细胞的自噬反应及其对脓毒症期间骨髓间充质干细胞功能的影响，需要 调查我们假设自噬是调节CO（离体） 通过MSC的细胞间作用和旁分泌作用增强MSC在脓毒症实验模型中的功能。到 为了验证我们的假设，我们提出了三个目标。在目标1中，我们将解读自噬的重要性， 在离体MSC的CO预处理后，通过C1-C4通路，在脓毒症期间增强MSC功能。MSC将 从Beclin 1 +/-和野生型（WT）小鼠收获，或Beclin 1将在WT MSC中沉默（shBeclin 1），和 然后在盲肠结扎和穿孔（CLP）（脓毒症的实验模型）后给予WT小鼠。我们将 确定CO预处理的增强功能是否在自噬蛋白缺陷的MSC中丧失。 我们还将研究CO对WT和自噬蛋白缺陷的MSC促进细菌增殖的作用。 清除、炎症消退、器官功能保护和存活。在目标2中，我们将确定 细胞外囊泡（EV）在用CO预处理的MSC的旁分泌作用中的作用，以及 研究自噬在这种反应中的重要性。EV将从来源于WT的MSC分离， Beclin 1 +/-小鼠，或shBeclin 1 MSC，并在败血症发作后给予WT小鼠。在目标3中，我们 确定用CO离体预处理MSC是否改善自噬蛋白的结果 在败血症期间缺乏的小鼠。将用CO预处理的WT MSC给予Beclin 1 +/-小鼠，并且 在CLP诱导的脓毒症后，自噬被抑制的小鼠。此外，我们将评估 来源于暴露于CO的MSC的EV对自噬蛋白缺陷小鼠的脓毒症结果的影响如果成功， 本申请将阐明MSC改善宿主对脓毒症应答的机制。阐明 自噬在MSC中的重要性，以及CO对MSC功能的增强也将促进我们的研究。 了解MSC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.