SUMMARY/ABSTRACT Mucosal surfaces are under constant challenge from exposure to environmental or microbial agents. However the exact nature of interactions between innate immune cells in the regulation of the mucosal barrier is still poorly understood. This proposal will incorporate a team of investigators with diverse and complementary expertise to investigate the mechanisms of innate immune cross-talk in the regulation of mucosal barrier function. The single layer of epithelial cells lining the GI tract is the most fundamental element of the mucosal barrier while macrophages play a key role in mucosal barrier homeostasis and in immunity to intestinal pathogens. Previous studies of communication between these intestinal epithelial cells (IEC) have focused on unidirectional macrophage regulation of epithelial function. However, we believe that this view is overly simplistic and that a more complex regulatory circuit exists. We hypothesize that bidirectional cross-talk circuits between epithelial cells and macrophages play a key role in both homeostatic regulation of epithelial permeability and macrophage polarization. Here, we will investigate the molecular nature of bidirectional cross- talk circuits between epithelial cells and macrophages with the overall objective to identify how the cell-intrinsic activity of an essential protein tyrosine phosphatase (TCPTP) regulates molecular changes in one cell type, which can in turn modify the functional capacity of the other. We will test our hypothesis in 3 Specific Aims. Aim 1 will address how TCPTP modulates the monocyte differentiation continuum and macrophage polarization status in mucosal homeostasis vs. local inflammation in the intestine. Aim 2 will utilize adoptive transfer experiments to identify how TCPTP deletion in macrophages vs. IEC modulates intestinal permeability and antimicrobial responsiveness of both cell types in vivo and in vitro. Aim 3 will identify molecular mechanisms by which TCPTP regulates how these cell types cross-communicate with each other. We have established novel mouse lines and in vitro co-culture model systems for this study. We will use these model systems in a series of innovative and established approaches, to allow us to mechanistically define phosphatase regulation of these fundamental interactions between macrophages and epithelial cells in the regulation of mucosal barrier function and macrophage polarization. In addition, we will identify if these cell- intrinsic phosphatase regulated crosstalk mechanisms apply across species by using mouse and human model systems. These experiments are foundationally linked to prior work but unequivocally represent an exciting new direction that synergizes the expertise of the investigative team. The results from these studies are poised to generate significant advances in our understanding of fundamental basic mechanisms in innate immunobiology and cellular crosstalk at mucosal surfaces.