Platelets are the cellular mediators of thrombosis, but they also have central roles in immune responses. Stimulated platelets release many soluble immune mediators that recruit and activate white blood cells. Beta-2 microglobulin (?2M) is a chaperone molecule essential for MHCI trafficking and stability. Platelets contain abundant ?2M that is released upon activation into the extracellular environment. Elevated plasma ?2M is associated with increased risk for inflammatory diseases, including cardiac events, and previous reports suggested it has direct signaling effects on monocytes. Through preliminary studies we have shown that ?2M directly promotes a pro-inflammatory response in monocytes. We have generated unique platelet specific ?2M-/- (Plt-?2M-/-) mice that we will use with a MI model to determine the mechanisms of ?2M regulated monocyte responses and their effect on inflammation and fibrosis. Using this novel mouse model, we have discovered that platelets are the major source of plasma ?2M and that it is significantly elevated post- myocardial infarction (MI). Work from our lab has shown that platelet-derived ?2M is critical to a pro- inflammatory (Ly6CHi) monocyte response post-MI. This lead us to hypothesize that platelet derived ?2M is a novel regulator of monocyte response to MI. To pursue this hypothesis, I propose the following Aims: 1) To demonstrate mechanisms of platelet mediated monocyte inflammatory phenotype, 2) To demonstrate how platelet-derived ?2M mediates monocyte responses and outcomes in an ischemic myocardial injury model. We will perform studies using primary mouse and human monocytes as well as a human monocytic cell-line (THP-1) to demonstrate the signaling mechanisms of ?2M related monocyte polarization. Monocyte phenotype will be characterized by surface markers using flow cytometry and cytokines using ELISA. We will determine the mechanism of ?2M-induced monocyte activation through use of surface plasmon resonance and receptor and signal transduction inhibitors, confirmed by immunoblot. Heart function and MI responses in our WT and Plt-?2M-/- mice will be analyzed using echocardiogram and histology. Plasma will be collected to determine the cytokine profile of our mouse models. Hearts will be collected for qRT-PCR and single cell isolation to phenotype the macrophages and fibrotic responses to MI. These data will define novel non-chaperone, direct inflammatory roles for ?2M in monocyte and subsequent macrophage responses to MI.
Due to modern treatments, the number of people who suffer heart attacks has steadily declined; however, the number of people who suffer heart failure after a heart attack has risen. This conflicting phenomenon highlights the need to understand novel mechanisms of heart repair after a heart attack for potential therapeutic targets. Our group intends to understand a unique way that platelets help white blood cells participate in the healing of the heart after a heart attack.
