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

描述（由申请人提供）： 由Manoj Pillai博士提出的R 03应用程序旨在完成、测试和扩展在K 08 DK 073701支持下生成的模型系统。最初的K 08奖专注于骨髓微环境中巨噬细胞基质细胞相互作用的作用。以前，我们已经表明巨噬细胞-基质的相互作用是重要的，在生产几个因素，这是已知的重要监管机构在造血ME，因为它是在体外测定。体外数据与临床观察结果的相关性表明了体内相关性。然而，这些模型未能证明因果关系。为了解决这一限制，巨噬细胞特异性，四环素诱导的转基因小鼠（CD 68-rtTA）已创建和表征。这种转基因小鼠可用于表达可以以可逆方式增加或抑制巨噬细胞的转基因，从而提供直接测量其功能结果的方法。目前正在制备另外的相关转基因和报告基因构建体。我们建议通过使用该模型来测试该模型，以确定在注射间充质基质细胞（MSC）后报告的组织再生是否需要驻留的巨噬细胞。 MSC被定义为间充质来源的细胞，能够分化成包括骨、软骨和脂肪组织的几种谱系。最近有显着的兴趣在使用MSC的组织再生和修复在不同的设置-心肌梗死，糖尿病，肝损伤等，但是，MSC的假定的有益作用的生物学机制仍然未知。鉴于MSC从靶组织的恢复通常是低的和短暂的，输注的MSC不太可能直接有助于修复受损组织。我们推测MSC对组织再生的有益作用可能是由MSC相互作用的常驻细胞介导的。已知单核细胞/巨噬细胞归巢于各种组织，并被MSC信号修饰;因此我们进一步假设它们在MSC介导的有益作用中发挥作用。提出了两个具体目标来检验这一假设。在第一个目标中，将测试MSC在致命辐射后拯救小鼠的能力。对照动物将不接受任何MSC支持。在第二个目的中，在MSC输注之前和之后的不同时间点，巨噬细胞将在CD 68-rtTA小鼠中被消除或抑制。这将使我们能够确定MSC输注后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，本R 03中的研究将扩展和测试K 08支持开发的模型。两个指导奖的结果将提供R 01申请所需的初步数据，这反过来又将促进PI向独立研究者的过渡。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种治疗方法的益处至关重要。 公共卫生相关性：允许诱导和可逆的增强或抑制巨噬细胞功能的转基因小鼠模型的开发将允许直接评估巨噬细胞在各种微环境中发挥的作用。它还可以用于研究MSC引起组织再生的机制，这对于优化这种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.