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A2469 - The Circadian Clock in Myeloid Cells Regulates the Severity of Ventilator-Induced Lung Injury in Mice
Author Block: M. Felten1, L. G. Teixeira-Alves1, E. Letsiou1, C. Chaput1, H. C. Muller-Redetzky1, N. W. Suttorp1, A. Kramer2, B. Maier2, M. Witzenrath1; 1Department of Infectious Diseases and Pulmonary Medicine, Charite, Berlin, Germany, 2Department of Clinical Immunology - Chronobiology, Charite, Berlin, Germany.
Introduction. Mechanical ventilation (MV) is a life-saving intervention in intensive care units (ICU), but may also exacerbate pre-existing lung injury. Ventilator-induced lung injury (VILI), is characterized by an excessive release of inflammatory cytokines, neutrophil-predominant inflammation and increased pulmonary permeability. Interestingly, circadian rhythm (CR) has emerged as important modulator of pulmonary immune responses and might critically influence outcome of ICU patients. Recently, we demonstrated for the first time that the severity of VILI is modulated by CR variations. In this study, we are focusing on exploring the role of the internal clock within neutrophils in VILI.
Methods. To explore whether the influence of CR on VILI is due to a cell-specific internal clock, we employed mice lacking the core clock gene Bmal1 in myeloid cells (LyzMCre-Bmal1-/-) in constant darkness. LyzMCre mice were used as controls. Mice were subjected to high tidal volume MV (HVt:34ml/kg, Inspiration:Expiration(I:E)-Ratio 1:1, 4h) to induce VILI. MV was initiated at circadian time (CT) 0 or CT12. Lung compliance, pulmonary permeability, neutrophil recruitment, and markers of pulmonary inflammation were measured to quantify VILI. To further analyze neutrophil specific inflammatory responses in vitro, myeloid cells from bone marrow of WT and Bmal1-deficient animals were isolated at dawn ZT0 (Zeitgeber time 0) and dusk (ZT12), incubated with DCFH-DA (2′,7′-Dichlorofluorescin diacetate) and stimulated for 15min with H2O2, PMA or PBS as control. Neutrophil activation (Ly6G/CD11b surface expression) and ROS production (intracellular DCFH-DA/Ly6G+ cells) was measured via flow cytometry.
Results. In vivo, injurious ventilation of control mice at CT0 led to a significant worsening of oxygenation, decrease of pulmonary compliance, and increased mortality compared to CT12. Interestingly, LyzMCre-Bmal1-/- mice did not exhibit any significant differences when subjected to MV at CT0 or CT12. Moreover, mortality in LyzMCre-Bmal1-/- mice was significantly reduced compared to LyzMCre controls (CT0). In vitro, neutrophils isolated from control (LyzMCre) mice at ZT0 showed a significantly higher level of baseline activation and increased ROS production after PMA-stimulation compared to neutrophils isolated at ZT12. There were no differences in ROS production from LyzMCre-Bmal1-/- neutrophils isolated at ZT0 compared to ZT12.
Conclusion. Our results show that deficiency of the clock gene Bmal1 in myeloid cells leads to increased survival after injurious ventilation in vivo and loss of circadian variations in ROS production from neutrophils in vitro. These data suggest that the internal clock in myeloid cells is an important modulator of VILI severity and might serve as potential therapeutic target.