Ventilator-induced Lung Injury Is Modulated by the Circadian Clock.

Rationale: Mechanical ventilation (MV) is life-saving but may evoke ventilator-induced lung injury (VILI). Objectives: To explore how the circadian clock modulates severity of murine VILI via the core clock component BMAL1 (basic helix-loop-helix ARNT like 1) in myeloid cells. Methods: Myeloid cell BMAL1-deficient (LysM (lysozyme 2 promoter/enhancer driving cre recombinase expression)Bmal1-/-) or wild-type control (LysMBmal1+/+) mice were subjected to 4 hours MV (34 ml/kg body weight) to induce lung injury. Ventilation was initiated at dawn or dusk or in complete darkness (circadian time [CT] 0 or CT12) to determine diurnal and circadian effects. Lung injury was quantified by lung function, pulmonary permeability, blood gas analysis, neutrophil recruitment, inflammatory markers, and histology. Neutrophil activation and oxidative burst were analyzed ex vivo. Measurements and Main Results: In diurnal experiments, mice ventilated at dawn exhibited higher permeability and neutrophil recruitment compared with dusk. Experiments at CT showed deterioration of pulmonary function, worsening of oxygenation, and increased mortality at CT0 compared with CT12. Wild-type neutrophils isolated at dawn showed higher activation and reactive oxygen species production compared with dusk, whereas these day-night differences were dampened in LysMBmal1-/- neutrophils. In LysMBmal1-/- mice, circadian variations in VILI severity were dampened and VILI-induced mortality at CT0 was reduced compared with LysMBmal1+/+ mice. Conclusions: Inflammatory response and lung barrier dysfunction upon MV exhibit diurnal variations, regulated by the circadian clock. LysMBmal1-/- mice are less susceptible to ventilation-induced pathology and lack circadian variation of severity compared with LysMBmal1+/+ mice. Our data suggest that the internal clock in myeloid cells is an important modulator of VILI.

CD169/SIGLEC1 is expressed on circulating monocytes in COVID-19 and expression levels are associated with disease severity.

Coronavirus disease 2019 (COVID-19) is caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Type I interferons are important in the defense of viral infections. Recently, neutralizing IgG auto-antibodies against type I interferons were found in patients with severe COVID-19 infection. Here, we analyzed expression of CD169/SIGLEC1, a well described downstream molecule in interferon signaling, and found increased monocytic CD169/SIGLEC1 expression levels in patients with mild, acute COVID-19, compared to patients with severe disease. We recommend further clinical studies to evaluate the value of CD169/SIGLEC1 expression in patients with COVID-19 with or without auto-antibodies against type I interferons.

Ventilator-induced lung injury is aggravated by antibiotic mediated microbiota depletion in mice.

BACKGROUND: Antibiotic exposure alters the microbiota, which can impact the inflammatory immune responses. Critically ill patients frequently receive antibiotic treatment and are often subjected to mechanical ventilation, which may induce local and systemic inflammatory responses and development of ventilator-induced lung injury (VILI). The aim of this study was to investigate whether disruption of the microbiota by antibiotic therapy prior to mechanical ventilation affects pulmonary inflammatory responses and thereby the development of VILI. METHODS: Mice underwent 6-8 weeks of enteral antibiotic combination treatment until absence of cultivable bacteria in fecal samples was confirmed. Control mice were housed equally throughout this period. VILI was induced 3 days after completing the antibiotic treatment protocol, by high tidal volume (HTV) ventilation (34 ml/kg; positive end-expiratory pressure = 2 cmH2O) for 4 h. Differences in lung function, oxygenation index, pulmonary vascular leakage, macroscopic assessment of lung injury, and leukocyte and lymphocyte differentiation were assessed. Control groups of mice ventilated with low tidal volume and non-ventilated mice were analyzed accordingly. RESULTS: Antibiotic-induced microbiota depletion prior to HTV ventilation led to aggravation of VILI, as shown by increased pulmonary permeability, increased oxygenation index, decreased pulmonary compliance, enhanced macroscopic lung injury, and increased cytokine/chemokine levels in lung homogenates. CONCLUSIONS: Depletion of the microbiota by broad-spectrum antibiotics prior to HTV ventilation renders mice more susceptible to developing VILI, which could be clinically relevant for critically ill patients frequently receiving broad-spectrum antibiotics.

Antihistone Properties of C1 Esterase Inhibitor Protect against Lung Injury.

RATIONALE: Acute respiratory distress syndrome is characterized by alveolar epithelial cell injury, edema formation, and intraalveolar contact phase activation. OBJECTIVES: To explore whether C1 esterase inhibitor (C1INH), an endogenous inhibitor of the contact phase, may protect from lung injury in vivo and to decipher the possible underlying mechanisms mediating protection. METHODS: The ability of C1INH to control the inflammatory processes was studied in vitro and in vivo. MEASUREMENTS AND MAIN RESULTS: Here, we demonstrate that application of C1INH alleviates bleomycin-induced lung injury via direct interaction with extracellular histones. In vitro, C1INH was found to bind all histone types. Interaction with histones was independent of its protease inhibitory activity, as demonstrated by the use of reactive-center-cleaved C1INH, but dependent on its glycosylation status. C1INH sialylated-N- and -O-glycans were not only essential for its interaction with histones but also to protect against histone-induced cell death. In vivo, histone-C1INH complexes were detected in bronchoalveolar lavage fluid from patients with acute respiratory distress syndrome and multiple models of lung injury. Furthermore, reactive-center-cleaved C1INH attenuated pulmonary damage evoked by intravenous histone instillation. CONCLUSIONS: Collectively, C1INH administration provides a new therapeutic option for disorders associated with histone release.

Increasing the inspiratory time and I:E ratio during mechanical ventilation aggravates ventilator-induced lung injury in mice.

INTRODUCTION: Lung-protective ventilation reduced acute respiratory distress syndrome (ARDS) mortality. To minimize ventilator-induced lung injury (VILI), tidal volume is limited, high plateau pressures are avoided, and positive end-expiratory pressure (PEEP) is applied. However, the impact of specific ventilatory patterns on VILI is not well defined. Increasing inspiratory time and thereby the inspiratory/expiratory ratio (I:E ratio) may improve oxygenation, but may also be harmful as the absolute stress and strain over time increase. We thus hypothesized that increasing inspiratory time and I:E ratio aggravates VILI. METHODS: VILI was induced in mice by high tidal-volume ventilation (HVT 34 ml/kg). Low tidal-volume ventilation (LVT 9 ml/kg) was used in control groups. PEEP was set to 2 cm H2O, FiO2 was 0.5 in all groups. HVT and LVT mice were ventilated with either I:E of 1:2 (LVT 1:2, HVT 1:2) or 1:1 (LVT 1:1, HVT 1:1) for 4 hours or until an alternative end point, defined as mean arterial blood pressure below 40 mm Hg. Dynamic hyperinflation due to the increased I:E ratio was excluded in a separate group of animals. Survival, lung compliance, oxygenation, pulmonary permeability, markers of pulmonary and systemic inflammation (leukocyte differentiation in lung and blood, analyses of pulmonary interleukin-6, interleukin-1ß, keratinocyte-derived chemokine, monocyte chemoattractant protein-1), and histopathologic pulmonary changes were analyzed. RESULTS: LVT 1:2 or LVT 1:1 did not result in VILI, and all individuals survived the ventilation period. HVT 1:2 decreased lung compliance, increased pulmonary neutrophils and cytokine expression, and evoked marked histologic signs of lung injury. All animals survived. HVT 1:1 caused further significant worsening of oxygenation, compliance and increased pulmonary proinflammatory cytokine expression, and pulmonary and blood neutrophils. In the HVT 1:1 group, significant mortality during mechanical ventilation was observed. CONCLUSION: According to the "baby lung" concept, mechanical ventilation-associated stress and strain in overinflated regions of ARDS lungs was simulated by using high tidal-volume ventilation. Increase of inspiratory time and I:E ratio significantly aggravated VILI in mice, suggesting an impact of a "stress/strain × time product" for the pathogenesis of VILI. Thus increasing the inspiratory time and I:E ratio should be critically considered.

Dynamics of pulmonary endothelial barrier function in acute inflammation: mechanisms and therapeutic perspectives.

The lungs provide a large inner surface to guarantee respiration. In lung alveoli, a delicate membrane formed by endo- and epithelial cells with their fused basal lamina ensures rapid and effective gas exchange between alveolar and vascular compartments while concurrently forming a robust barrier against inhaled particles and microbes. However, upon infectious or sterile inflammatory stimulation, tightly regulated endothelial barrier leakiness is required for leukocyte transmigration. Further, endothelial barrier disruption may result in uncontrolled extravasation of protein-rich fluids. This brief review summarizes some important mechanisms of pulmonary endothelial barrier regulation and disruption, focusing on the role of specific cell populations, coagulation and complement cascades and mediators including angiopoietins, specific sphingolipids, adrenomedullin and reactive oxygen and nitrogen species for the regulation of pulmonary endothelial barrier function. Further, current therapeutic perspectives against development of lung injury are discussed.

Mechanical ventilation drives pneumococcal pneumonia into lung injury and sepsis in mice: protection by adrenomedullin.

INTRODUCTION: Ventilator-induced lung injury (VILI) contributes to morbidity and mortality in acute respiratory distress syndrome (ARDS). Particularly pre-injured lungs are susceptible to VILI despite protective ventilation. In a previous study, the endogenous peptide adrenomedullin (AM) protected murine lungs from VILI. We hypothesized that mechanical ventilation (MV) contributes to lung injury and sepsis in pneumonia, and that AM may reduce lung injury and multiple organ failure in ventilated mice with pneumococcal pneumonia. METHODS: We analyzed in mice the impact of MV in established pneumonia on lung injury, inflammation, bacterial burden, hemodynamics and extrapulmonary organ injury, and assessed the therapeutic potential of AM by starting treatment at intubation. RESULTS: In pneumococcal pneumonia, MV increased lung permeability, and worsened lung mechanics and oxygenation failure. MV dramatically increased lung and blood cytokines but not lung leukocyte counts in pneumonia. MV induced systemic leukocytopenia and liver, gut and kidney injury in mice with pneumonia. Lung and blood bacterial burden was not affected by MV pneumonia and MV increased lung AM expression, whereas receptor activity modifying protein (RAMP) 1-3 expression was increased in pneumonia and reduced by MV. Infusion of AM protected against MV-induced lung injury (66% reduction of pulmonary permeability p < 0.01; prevention of pulmonary restriction) and against VILI-induced liver and gut injury in pneumonia (91% reduction of AST levels p < 0.05, 96% reduction of alanine aminotransaminase (ALT) levels p < 0.05, abrogation of histopathological changes and parenchymal apoptosis in liver and gut). CONCLUSIONS: MV paved the way for the progression of pneumonia towards ARDS and sepsis by aggravating lung injury and systemic hyperinflammation leading to liver, kidney and gut injury. AM may be a promising therapeutic option to protect against development of lung injury, sepsis and extrapulmonary organ injury in mechanically ventilated individuals with severe pneumonia.

Adjunctive therapy with the Tie2 agonist Vasculotide reduces pulmonary permeability in Streptococcus pneumoniae infected and mechanically ventilated mice.

Community acquired pneumonia, mainly caused by Streptococcus pneumoniae (S.pn.), is a common cause of death worldwide. Despite adequate antibiotic therapy, pneumococcal pneumonia can induce pulmonary endothelial hyperpermeability leading to acute lung injury, which often requires mechanical ventilation (MV) causing ventilator-induced lung injury (VILI). Endothelial stabilization is mediated by angiopoietin-1 induced Tie2 activation. PEGylated (polyethylene glycol) Tie2-agonist Vasculotide (VT) mimics Angiopietin-1 effects. Recently, VT has been shown to reduce pulmonary hyperpermeability in murine pneumococcal pneumonia. The aim of this study was to determine whether VT reduces lung damage in S.pn. infected and mechanically ventilated mice. Pulmonary hyperpermeability, immune response and bacterial load were quantified in S.pn. infected mice treated with Ampicillin + /-VT and undergoing six hours of MV 24 h post infection. Histopathological lung changes, Tie2-expression and -phosphorylation were evaluated. VT did not alter immune response or bacterial burden, but interestingly combination treatment with ampicillin significantly reduced pulmonary hyperpermeability, histological lung damage and edema formation. Tie2-mRNA expression was reduced by S.pn. infection and/or MV but not restored by VT. Moreover, Tie2 phosphorylation was not affected by VT. These findings indicate that VT may be a promising adjunctive treatment option for prevention of VILI in severe pneumococcal pneumonia.

Neutrophil-Derived Extracellular Vesicles Activate Platelets after Pneumolysin Exposure.

Pneumolysin (PLY) is a pore-forming toxin of Streptococcus pneumoniae that contributes substantially to the inflammatory processes underlying pneumococcal pneumonia and lung injury. Host responses against S. pneumoniae are regulated in part by neutrophils and platelets, both individually and in cooperative interaction. Previous studies have shown that PLY can target both neutrophils and platelets, however, the mechanisms by which PLY directly affects these cells and alters their interactions are not completely understood. In this study, we characterize the effects of PLY on neutrophils and platelets and explore the mechanisms by which PLY may induce neutrophil-platelet interactions. In vitro studies demonstrated that PLY causes the formation of neutrophil extracellular traps (NETs) and the release of extracellular vesicles (EVs) from both human and murine neutrophils. In vivo, neutrophil EV (nEV) levels were increased in mice infected with S. pneumoniae. In platelets, treatment with PLY induced the cell surface expression of P-selectin (CD62P) and binding to annexin V and caused a significant release of platelet EVs (pl-EVs). Moreover, PLY-induced nEVs but not NETs promoted platelet activation. The pretreatment of nEVs with proteinase K inhibited platelet activation, indicating that the surface proteins of nEVs play a role in this process. Our findings demonstrate that PLY activates neutrophils and platelets to release EVs and support an important role for neutrophil EVs in modulating platelet functions in pneumococcal infections.