Role of macrophages in MSC-mediated recovery of hematopoiesis after irradiation

描述(由申请人提供)： 这个R03应用程序由Manoj Pillai博士提出，旨在完成、测试和扩展在K08 DK073701支持下生成的模型系统。最初的K08奖侧重于巨噬细胞基质细胞相互作用在骨髓微环境中的作用。在此之前，我们已经证明巨噬细胞-间质的相互作用在产生几种因子方面是重要的，这些因子在体外测定的造血ME中被认为是重要的调节因子。体外数据与临床观察的相关性表明了体内的相关性。然而，这些模型不能证明因果关系。为了解决这一局限性，建立了一种巨噬细胞特异性的、四环素诱导的转基因小鼠(CD68-RTTA)并进行了鉴定。这种转基因小鼠可以用来表达可以以可逆方式增强或抑制巨噬细胞的转基因，从而提供了一种直接测量其功能结果的方法。目前，正在准备更多相关的转基因和报告基因构建。我们建议测试这个模型，用它来确定在注射间充质基质细胞(MSC)后，是否需要常驻巨噬细胞来进行组织再生。 MSC被定义为起源于间充质的细胞，能够分化为几个谱系，包括骨、软骨和脂肪组织。近年来，在心肌梗死、糖尿病、肝损伤等不同的环境中，使用MSC进行组织再生和修复已经引起了人们的极大兴趣。然而，MSC潜在的有益作用的生物学机制仍然不清楚。鉴于MSC从靶组织中的恢复通常是低和短暂的，因此，注入的MSC不太可能直接有助于受损组织的修复。我们推测，MSC对组织再生的有益影响可能是通过与MSC相互作用的驻留细胞介导的。单核/巨噬细胞是已知的多种组织的宿主，并被MSC信号修饰；因此，我们进一步假设它们在MSC介导的有益效应中发挥作用。为了检验这一假说，本文提出了两个具体目标。在第一个目标中，将测试MSC在致命辐射后拯救老鼠的能力。对照动物将不会得到任何MSC支持。在第二个目标中，CD68-RTTA小鼠体内的巨噬细胞在输注MSC之前和之后的不同时间点将被清除或抑制。这将使我们能够确定输注MSC后的造血恢复是否需要巨噬细胞，以及发生这种情况的时间间隔。因此，R03中的研究将扩展和测试由K08支持开发的模型。这两个指导性奖项的结果将提供R01申请所需的初步数据，这反过来将促进PI向独立调查人员的过渡。此外，了解MSC介导的再生的精确生物学机制对于最终优化这一治疗方法的好处将是至关重要的。 与公共卫生相关：发展转基因小鼠模型，允许诱导和可逆地增强或抑制巨噬细胞的功能，将允许直接评估巨噬细胞在各种微环境中所起的作用。它也可以被用来研究MSC促进组织再生的机制，这对于优化这一有前途的治疗模式至关重要。

DESCRIPTION (provided by applicant): This R03 application, proposed by Dr. Manoj Pillai, seeks to complete, test, and expand a model system generated with support from K08 DK073701. The original K08 award focused on the role of macrophage stromal cell interactions in the marrow microenvironment. Previously we have shown macrophage-stromal interactions to be important in producing several factors which are known to be important regulators in the hematopoietic ME as it is assayed in vitro. Correlations of in vitro data with clinical observations have suggested in vivo relevance. However these models fall short of proving cause and effect. To address this limitation, a macrophage-specific, tetracycline-inducible transgenic mouse (CD68-rtTA) has been created and characterized. This transgenic mouse can be used to express transgenes that can either augment or inhibit macrophages in a reversible manner, thereby providing a means to directly measure the outcome of their function. Currently additional relevant transgenes and reporter constructs are being prepared. We propose to test this model by using it to determine if resident macrophages are required for the tissue regeneration that is reported following injection of Mesenchymal Stromal Cells (MSC). MSC are defined as cells of mesenchymal origin, capable of differentiation in to several lineages including bone, cartilage and adipose tissue. There has been significant interest recently in using MSC for tissue regeneration and repair in diverse settings - myocardial infarction, diabetes mellitus, liver injury etc. However, the biologic mechanisms underlying the putative beneficial effects of MSC remain unknown. Given that the recovery of MSC from target tissue is typically low and transient, it is unlikely that the infused MSC contribute directly to repair of the damaged tissues. We hypothesize that the beneficial effects of MSC on tissue regeneration may be mediated by resident cells with which the MSC interact. Monocytes/ macrophages are known to home to a variety of tissues, and are modified by MSC signals; hence we further hypothesize that they play a role in the beneficial effects mediated by MSC. Two specific aims are proposed to test this hypothesis. In the first aim, MSC will be tested for their ability to rescue mice after lethal radiation. Control animals will not receive any MSC support. In the second aim, macrophages will be eliminated or inhibited in the CD68-rtTA mouse prior to, and at different time points following MSC infusion. This will allow us to determine if hematopoeitic recovery after MSC infusion requires macrophages, and the time interval in which this occurs. Hence the studies in this R03 will expand and test the models developed by the K08 support. Results from both mentored awards will provide preliminary data needed for an R01 application, which in turn will facilitate the PI's transition to an independent investigator. Furthermore, understanding the precise biologic mechanisms of MSC-mediated regeneration will be critical for eventually optimizing the benefits of this therapeutic approach. PUBLIC HEALTH RELEVANCE: The development of a transgenic mouse model that allows for the inducible and reversible augmentation or inhibition of macrophage function will allow for a direct assessment of the role macrophages play in various microenvironments. It can also be exploited to study the mechanisms by which MSC bring about tissue regeneration, which is critical for optimizing this promising mode of therapy.