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.