Summary/Abstract The overall goal of this proposal is to better understand the role of the tyrosine kinase receptor EphA2 signaling pathway in alveolar macrophages during Pneumocystis pneumonia (PCP). Pneumocystis jirovecii pneumonia (PJP) remains a significant cause of morbidity and mortality in AIDS1,2. During AIDS and other immunosuppressive states, the absence of CD4 lymphocytic immunity results in exuberant and often fatal PJP3,4. Myeloid cells, particularly alveolar macrophages (AMs) are crucial for anti-PJP innate immunity5-8. The binding of Pneumocystis to alveolar macrophages mediates early host immune response to the fungus9,10. AMs further promote killing and clearance of organisms but are also major sources of proinflammatory mediators contributing to profound pulmonary inflammation and lung injury during PJP11-13. To date, extremely little is known regarding the role of EphA2 receptor and Pneumocystis engagement in mediating subsequent effects on the host immune response and fungal killing during Pneumocystis pneumonia (PCP). We have previously shown the importance of the β-glucan receptor EphA2 in Pneumocystis organism attachment to lung epithelial cells14. Furthermore, we demonstrated that following EphA2 receptor-ligand engagement with Pneumocystis β-glucan carbohydrates, the tyrosine kinase receptor is activated in epithelial cells14. Now, our preliminary studies further indicate the importance of the EphA2 receptors on AMs in inflammatory responses to Pneumocystis. To date, there are no published descriptions of the potential roles of EphA2 on AMs during fungal infection pathogenesis. We now provide exciting new initial in vivo data demonstrating that EphA2-deficient mice have significantly less proinflammatory responses and significantly greater organism burden in both immunocompromised and immunocompetent mouse models of PCP. We therefore hypothesize that EphA2 receptors signal following Pneumocystis binding to alveolar macrophages mediating early host immune recognition and response to the organism. In addition, based on our preliminary in vivo PCP model data, we further hypothesize that EphA2-receptor signaling in AMs is critical for mounting proper lung inflammation and organism control during PCP. Two Specific Aims are proposed. Aim 1: We will characterize the function of EphA2 signaling pathways in AMs following in vitro organism attachment. We will specifically study the resulting cytokine response in AMs, as well as subsequent organism killing by AMs. To address this, we will examine the function of EphA2 in AMs derived from EphA2 (EphA2-/-) receptor knockout and wildtype mice challenged with mouse derived Pneumocystis murina. Specifically, we will study the binding kinetics and inflammatory responses following P. murina interactions with AMs. Initial data from our lab suggests significantly less inflammatory cytokine responses in macrophages from EphA2-/- versus wildtype controls. Furthermore, we will analyze the up...