Saturday, April 27, 2024
4/27/2024
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 ß-glucan receptor EphA2 in Pneumocystis organism attachment to lung epithelial cells14. Furthermore, we demonstrated that following EphA2 receptor-ligand engagement with Pneumocystis ß-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 uptake and killing of P. murina by macrophages, as well as cytokine release by these cells challenged with either P. murina as well as isolated Pneumocystis organism cell wall components. Aim 2: We will further determine the role of EphA2-signaling on modulating lung inflammation, AM subset recruitment, and organism burdens in the PCP immunocompetent and immunosuppressed models in mice. Our preliminary studies demonstrate that CD4-depleted EphA2-/- mice with PCP exhibit significantly increased organism burdens compared to their wildtype counterparts. Accordingly, we will evaluate the time course and magnitudes of organism clearance and inflammatory cytokines in wildtype and EphA2-/- mice. We will directly contrast these parameters in immune competent compared to CD4-depleted mice. Furthermore, we will directly isolate and assess macrophages derived from both groups of infected mice to assess their ability to kill Pneumocystis and generate adequate cytokine responses. We will also employ flow cytometry to further isolate and characterize distinct pulmonary macrophages subset recruitment. This will include whether the AMs are relatively Type 1 or Type 2 polarized and will further assess the recruitment of other macrophage populations include tissue-resident alveolar macrophages (TR-AMs), monocyte- derived alveolar macrophages (Mo-AMs), and interstitial macrophages (IMs). These determinations will be performed in both mouse strains tested in the PCP models proposed. These studies promise valuable insights into the role of EphA2 receptor signaling pathway in macrophages during PCP and should provide initial data that may inform future targeted therapies of this pathway for the treatment of PCP. We anticipate that these studies will define the importance of the of EphA2 receptor signaling pathway in organism killing and regulating the host response in PCP. Modulating these pathways may serve as a novel strategy for therapeutic intervention potentially beneficial to patients with PJP when provided in addition to traditional antibiotic agents.
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