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A7570 - Depletion of the Resident Alveolar Macrophage Pool Upon Influenza Virus Infection Is Death Ligand-Dependent and Contributes to Disease Severity
Author Block: C. Malainou, C. Peteranderl, W. Seeger, J. H. Lohmeyer, S. Herold; Department of Medicine II, Pulmonary and Critical Care Medicine and Infectious Diseases, Universities of Giessen and Marburg Lung Center, member of the German Center for Lung Research (DZL), Giessen, Germany.
RATIONALE: Resident alveolar macrophage (rAM) depletion has a major impact on disease progression in influenza virus (IV)- induced lung injury and acute respiratory distress syndrome (ARDS). Therefore, we aim to better understand the time course, cellular crosstalk and molecular pathways which cause rAM reprogramming and death, as improving rAM host defense function and survival poses a potential therapeutic target in order to ameliorate disease severity and prevent bacterial superinfection. METHODS: C57BL/6 wild-type mice were infected with influenza virus (IV) PR/8 by intra-tracheal application. Bronchoalveolar lavage (BAL) was harvested at different time points for flow cytometry (FACS) analysis. rAM death pathways were assessed by gene expression analysis (RT² ProfilerTM PCR mouse cell death PathwayFinder). Additionally, FACS was performed on rAM after ex vivo infection and treatment with BAL from infected mice. Apoptosis-inducing ligands of the tumor necrosis factor superfamily (TNFSF) were analyzed on gene expression level in primary isolated human rAM after ex vivo infection.RESULTS: Significant rAM depletion begins on day 3 post IV infection with rAM numbers reaching their lowest level on day 7, as shown by FACS analysis. Interestingly, FACS analyses revealed that although the majority of rAM was subjected to apoptotic death, the percentage of directly infected rAM by influenza virus was discordantly low. Gene expression analysis confirmed apoptosis being the main pathway inducing rAM death as indicated by significant upregulation of apoptosis-related factors such as Fas ligand, Caspase-9 and Cflar (CASP8 and FADD-like apoptosis regulator). Remarkably, treatment of rAM isolated from non-infected mice with day 7 BAL had a more prominent apoptotic effect than direct PR8 infection 24h after treatment. Further investigation of potential apoptosis triggering signals through gene expression analysis showed a significant upregulation of various TNFSF members, such as TNFSF 11, 14 and 18 in ex vivo IV-infected rAM.
CONCLUSIONS: IV infection of wild-type mice results in rapid apoptosis induction and rAM depletion. Extensive apoptosis despite low infection rates implies that not direct infection, but a cellular signaling network involving pro-apoptotic factors causes rAM death. In line, various members of the TNF superfamily were upregulated in rAM in IV infection. Dissecting the specific cellular interactions and blocking the underlying molecular signaling events will enable us to improve rAM function and survival, and ultimately to define novel therapeutic strategies to reach better outcome of IV-induced pneumonia and ARDS.