Chronic Toll-like Receptor Ligation and Hematopoietic Stem Cell Aging

描述(申请人提供)：尽管造血系统是所有干细胞驱动的组织中最具特点的；考虑到造血干细胞(HSC)在整个生命周期内的天文数字输出需求，该系统已成为衰老和再生医学研究的最佳目标。鉴于最近发现微生物Toll样受体(TLR)配体可以直接影响HSC的动态平衡，旨在保护HSC免受感染应激的研究正变得越来越重要。在这里，我们介绍了一种新的实验方法来确定慢性感染相关条件是否会加速HSC老化。通过使用已建立的模型，我们准备回答有关慢性TLR结扎对HSC衰老和长期生理的影响的新问题。实验计划有两个具体目标：(1)确定慢性刺激小鼠骨髓中HSC和原始祖细胞的数量、表型、分化潜能和细胞周期状态；(2)严格检测从慢性TLR配体刺激的小鼠中回收的HSC的完整性。我们的策略包括研究慢性TLR结扎对HSC老化的即时和长期影响。每日腹腔注射脂多糖或Pam3CSK4将被用来模拟慢性感染。我们将通过评估治疗期间和整个年龄加速条件下的骨髓群体动力学、表面表型和祖细胞周期状态来确定慢性TLR刺激是否改变了HSC的完整性。多色流式细胞术以及免疫印迹和RT-PCR等分子分析将揭示慢性TLR刺激是否会在衰老的HSC中诱导谱系偏见。F1杂交受体的新应用也将允许在连续移植期间精确量化HSC的健康状况。明确的培养体系将进一步评估HSC的分化潜力。慢性感染每年都需要在患者治疗方面做出重大努力。慢性感染相关缺陷是否会导致HSC老化加速，这是我们准备在这里概述的研究中解决的一个重要问题。由于骨髓干细胞耗尽而导致的免疫系统功能减弱是衰老的常见后果。正因为如此，即使是轻微的感染也会对老年或免疫功能低下的患者构成严重威胁。这里概述的研究提供了一种新的方法来确定保护骨髓干细胞免受感染和衰老的破坏的潜在方法。这些信息可能导致临床应用，通过在具有挑战性的环境中保持免疫系统的完整性来延长老年或免疫受损患者的生命。

DESCRIPTION (provided by applicant): Whereas the hematopoietic system is the most characterized of any stem-cell driven tissue; and given the astronomical output requirements of hematopoietic stem cells (HSC) over a lifespan, this system has become an optimal target for studies on aging and regenerative medicine. In light of the recent discovery that microbial toll-like receptor (TLR) ligands can directly impact HSC homeostasis, research aimed at protecting HSC from the stress of infection is becoming increasingly relevant. Here we introduce a new experimental approach to determine whether chronic infection-related conditions hasten HSC aging. Through the use of an established model, we are primed to answer new questions concerning the effects of chronic TLR ligation on the aging and long-term physiology of HSC. The experimental plan is outlined by two Specific Aims, which are:(1) Determine the numbers, phenotype, differentiation potential, and cell cycle status of HSC and primitive progenitors in chronically-stimulated marrow, and (2) Rigorously test the integrity of HSC recovered from chronic TLR ligand-stimulated mice. Our strategy consists of investigating both the immediate and long-term effects of chronic TLR ligation on HSC aging. Daily intraperitoneal injections with LPS or Pam3CSK4 will be used to model chronic infection. We will determine if chronic TLR stimulation alters HSC integrity by evaluating bone marrow population dynamics, surface phenotypes, and progenitor cell cycle status during treatment, and throughout age-accelerating conditions. Multi-color flow cytometry and molecular assays such as western blot and RT-PCR will reveal whether chronic TLR stimulation induces lineage biases in aging HSC. Novel application of F1 hybrid recipients will also allow precise quantification of HSC fitness during serial transplants. Well-defined culture systems will further assess HSC differentiation potential. Chronic infections require significant efforts in patient treatment each year. Whether chronic infection-related deficiencies lead to accelerated HSC aging is an important question we are poised to address in the studies outlined here. Diminished immune system function due to exhaustion of bone marrow stem cells is a common consequence of aging. Because of this, even mild infections pose a serious threat to elderly or immune-compromised patients. The studies outlined here present a new approach for identifying potential means of protecting bone marrow stem cells from the ravages of infection and aging. This information may lead to clinical applications that prolong life in aged or immune-compromised patients by preserving the integrity of the immune system during challenging circumstances.