Inflammation is both a cause and consequence of many serious and life-threatening diseases, including cancer, heart disease, Alzheimer's disease, arthritis, and infectious diseases. Inflammation is central to the appropriate repair and regeneration of damaged tissue from injury, infection, and trauma. Central to this repair process are macrophages and monocytes; their polarization, phenotype, and function are essential to driving inflammation and repair after injury. Although the CD9 tetraspanin is highly expressed in several functionally distinct populations of regenerative macrophages, its function is poorly understood, in part due to a lack of high- specificity biochemical tools to interrogate its function. CD9 is composed of one small and one large extracellular loop bearing many different epitopes. To assess CD9 function in inflammation, it would be highly useful to have molecules that can (a) block specific epitopes/extracellular regions of CD9; (b) completely block CD9 function (act as antagonists); or (c) activate CD9 (act as agonists). Here we propose to address this technical gap by developing antibody fragments that bind specifically to the different CD9 epitopes to assess CD9 function in inflammation. Building a platform that binds and/or blocks all CD9 epitopes is important since it will allow us to elucidate how each epitope affects downstream signaling and macrophage phenotypes. This proposal is based on the overall hypothesis that CD9 is an important driver of macrophage polarization and that therefore it drives inflammation and repair. To test this, we will identify, recombinantly express, and validate single-chain variable fragments (scFv) targeting different CD9 epitopes in Aim 1.
In Aim 2 we will examine CD9 function in a mouse model of wound repair using epitope-specific anti-CD9 scFvs.
The tetraspanin protein CD9 is highly expressed on regenerative macrophages but can also be found on endothelial cells, other immune cells, myoblasts, gametes and cancer cells. We propose to develop a novel biochemical platform composed of a set of CD9-targeting antibody fragments that will be epitope-specific and that will display distinct agonistic and antagonistic function for interrogating macrophage function. These comprehensive reagents will allow the systematic elucidation of CD9 function in many disciplines, may themselves be therapeutic reagents and will be useful for discovering new mechanisms and targets in inflammatory diseases.
