B3-Subproject

Development and function of respiratory macrophages and dendritic cell subsets during bacterial pneumonia (Hackstein / Bauer / Lohmeyer)

Bacterial infections are a leading cause of community acquired as well as nosocomial pneumonia with significant mortality. Lung resident dendritic cells (DC) and macrophages (MP) recognize bacterial pathogens, initiate immune responses and regulate inflammation. Several DC subsets, such as CD103+ and CD103- conventional migratory DCs (cDCs), monocyte-derived DCs (moDCs) and plasmacytoid DCs as well as functionally distinct lung MP populations have been described, but their precise contribution to the respiratory immune response during bacterial infections remains unresolved. In the first funding period we dissected quantitative and functional changes of major lung DC and MP subsets during bacterial pneumonia in mice by multicolor flow cytometry/cell sorting and developed selective cell ablation techniques for lung mononuclear phagocyte subsets. We identified a key role of alveolar epithelial cell released GM-CSF in lung immunity and discovered a novel function of the RNA editing enzyme Adenosine deaminase 1 acting on RNA (ADAR1) in lung DC/MP development by crossing ADAR1 floxed mice to CD11cCre or LysM-Cre mice. We will extend these studies to clarify mechanisms which shape the composition of the respiratory MP and DC subsets and determine their temporal and spatial contribution to bacterial clearance, inflammation and restoration of lung immune homeostasis as well as tissue integrity during bacterial pneumonia. To elucidate the observed role of the RNA editing enzyme ADAR1 in lung DC/MP development and function we will identify the relevant ADAR1 domain and characterize the ADAR1-edited target structures which regulate immune cell development. Accordingly, bone marrow from ADAR1floxed/CD11c-Cre mice will be reconstituted with retroviral or in vitro transcribed ADAR1 constructs (e.g. ADAR1 p150, ADAR1 p110, deaminase mutant) and comparative transcriptome/miRNA profiling of ADAR1-dependent vs. non ADAR1-dependent DC subsets, will be performed. We will further employ established conditional cell-specific ablation/adoptive cell transfer strategies as well as genetic cell fate tracing approaches in bone marrow chimera models to dissect the role of ADAR1 in DC/MP subset ontogeny and function under steady state and inflammatory conditions in the established Klebsiella pneumonia mouse model. Finally, flow cytometric protocols developed in the previous funding period for identification and sorting of DC/MP subsets with corresponding functional capacities as observed in the mouse model will be employed to systematically characterize clinical BALF samples originating from pneumonia patients (>250 samples biobank stored) in correlation with clinical data. We further plan to include BALF samples collected from pneumonia patients within a placebo controlled clinical proof-of-concept study currently under PEI/Ethics Committee evaluation addressing the role of aerosolized GM-CSF as adjunctive DC/MP targeting therapy in pneumonia.