Organ injury leads to both local and systemic responses that promote removal of damaged cells and stimulate surviving cells to reconstitute the normal architecture. Studies in rodent models of organ injury and in human biopsies have shown that macrophages are recruited after injury and are induced by signals in the injured organ to express distinct activation states including an initial proinflammatory (?M1?) phenotype followed by a proreparative (?M2?) phenotype that is believed to be important for normal repair. In cases where repair is delayed or incomplete, macrophages can adopt a late profibrotic phenotype that appears to cross-talk with fibroblasts/myofibroblasts and promote organ fibrosis. The proposed roles of proreparative macrophages in normal organ repair and profibrotic macrophages in progressive organ fibrosis make these cell types of great interest to the translational research community. However, genetic tools that precisely identify these functionally distinct macrophage activation states are currently not available, and thus the origin, fate and specific effects of proreparative and profibrotic macrophages are not well defined. This proposal has been developed to generate transgenic mouse strains in which either proreparative or profibrotic macrophages can be tracked using turboGFP expression, and fate mapped, depleted or genetically altered using Cre expression. To achieve this, we are utilizing a modular cloning system to generate multiple synthetic promoters, and screening these promoters in vitro to identify the enhancer/promoter combination that is most specifically and robustly expressed in the desired cell type and activation state. We have successfully identified a synthetic promoter that is expressed only in M2-activated macrophages in vitro, and under Aim1 we will use this promoter (named Dali) to generate the transgenic mouse and validate the selective expression of turboGFP and Cre in M2 macrophages at baseline and following organ injury in vivo.
Under Aim 2 we will design and screen a second synthetic promoter to be used for identifying profibrotic macrophages, and use this synthetic promoter to establish a second mouse strain for targeting profibrotic macrophages in vivo. These two novel genetic tools are anticipated to be highly specific and robust, and thus valuable for the research community to investigate macrophage function in organ injury and repair in vitro and in vivo, and to guide new therapeutic approaches to manipulate macrophage activation to promote repair and restoration of function after injury.
Any organ in a body can be injured with impaired function, and the body tackles injury by eliminating the damaged cells, reducing inflammation, resolving the injury, and replacing injured cells with new cells to restore function. We and others have identified macrophages as a prominent regulator of this repair process. Our current proposal is to design and generate new genetic tools to better understand how macrophages change their fates in order to help restore organ function, which could provide guidance for future therapy.