Kenji Wakabayashi

Volutrauma, but not atelectrauma, induces systemic cytokine production by lung-marginated monocytes.

Objectives:Ventilator-induced lung injury has substantive impact on mortality of patients with acute respiratory distress syndrome. Although low tidal volume ventilation has been shown to reduce mortality, clinical benefits of open-lung strategy are controversial. In this study, we investigated the impact of two distinct forms of ventilator-induced lung injury, i.e., volutrauma and atelectrauma, on the progression of lung injury and inflammation, in particular alveolar and systemic cytokine production. Design:Ex vivo study. Setting:University research laboratory. Subjects:C57BL/6 mice. Interventions:Isolated, buffer-perfused lungs were allocated to one of three ventilatory protocols for 3 hours: control group received low tidal volume (7 mL/kg) with positive end-expiratory pressure (5 cm H2O) and regular sustained inflation; high-stretch group received high tidal volume (30–32 mL/kg) with positive end-expiratory pressure (3 cm H2O) and sustained inflation; and atelectasis group received the same tidal volume as control but neither positive end-expiratory pressure nor sustained inflation. Measurements and Main Results:Both injurious ventilatory protocols developed comparable levels of physiological injury and pulmonary edema, measured by respiratory system mechanics and lavage fluid protein. High-stretch induced marked increases in proinflammatory cytokines in perfusate and lung lavage fluid, compared to control. In contrast, atelectasis had no effect on perfusate cytokines compared to control but did induce some up-regulation of lavage cytokines. Depletion of monocytes marginated within the lung microvasculature, achieved by pretreating mice with IV liposome-encapsulated clodronate, significantly attenuated perfusate cytokine levels, especially tumor necrosis factor, in the high-stretch, but not atelectasis group. Conclusions:Volutrauma (high-stretch), but not atelectrauma (atelectasis), directly activates monocytes within the pulmonary vasculature, leading to cytokine release into systemic circulation. We postulate this as a potential explanation why open-lung strategy has limited mortality benefits in ventilated critically ill patients.

Influence of glutathione- S -transferase (GST) inhibition on lung epithelial cell injury: role of oxidative stress and metabolism

Oxidant-mediated tissue injury is key to the pathogenesis of acute lung injury. Glutathione-S-transferases (GSTs) are important detoxifying enzymes that catalyze the conjugation of glutathione with toxic oxidant compounds and are associated with acute and chronic inflammatory lung diseases. We hypothesized that attenuation of cellular GST enzymes would augment intracellular oxidative and metabolic stress and induce lung cell injury. Treatment of murine lung epithelial cells with GST inhibitors, ethacrynic acid (EA), and caffeic acid compromised lung epithelial cell viability in a concentration-dependent manner. These inhibitors also potentiated cell injury induced by hydrogen peroxide (H2O2), tert-butyl-hydroperoxide, and hypoxia and reoxygenation (HR). SiRNA-mediated attenuation of GST-π but not GST-μ expression reduced cell viability and significantly enhanced stress (H2O2/HR)-induced injury. GST inhibitors also induced intracellular oxidative stress (measured by dihydrorhodamine 123 and dichlorofluorescein fluorescence), caused alterations in overall intracellular redox status (as evidenced by NAD(+)/NADH ratios), and increased protein carbonyl formation. Furthermore, the antioxidant N-acetylcysteine completely prevented EA-induced oxidative stress and cytotoxicity. Whereas EA had no effect on mitochondrial energetics, it significantly altered cellular metabolic profile. To explore the physiological impact of these cellular events, we used an ex vivo mouse-isolated perfused lung model. Supplementation of perfusate with EA markedly affected lung mechanics and significantly increased lung permeability. The results of our combined genetic, pharmacological, and metabolic studies on multiple platforms suggest the importance of GST enzymes, specifically GST-π, in the cellular and whole lung response to acute oxidative and metabolic stress. These may have important clinical implications.

Sources of alveolar soluble TNF receptors during acute lung injury of different etiologies

Elevated soluble tumor necrosis factor-α receptor (sTNFR) levels in bronchoalveolar lavage fluid (BALF) are associated with poor patient outcome in acute lung injury (ALI). The mechanisms underlying these increases are unknown, but it is possible that pulmonary inflammation and increased alveolar epithelial permeability may individually contribute. We investigated mechanisms of elevated BALF sTNFRs in two in vivo mouse models of ALI. Anesthetized mice were challenged with intratracheal lipopolysaccharide or subjected to injurious mechanical ventilation. Lipopolysaccharide instillation produced acute intra-alveolar inflammation, but minimal alveolar epithelial permeability changes, with increased BALF sTNFR p75, but not p55. Increased p75 levels were markedly attenuated by alveolar macrophage depletion. In contrast, injurious ventilation induced substantial alveolar epithelial permeability, with increased BALF p75 and p55, which strongly correlated with total protein. BALF sTNFRs were not increased in isolated buffer-perfused lungs (devoid of circulating sTNFRs) subjected to injurious ventilation. These results suggest that lipopolysaccharide-induced intra-alveolar inflammation upregulates alveolar macrophage-mediated production of sTNFR p75, whereas enhanced alveolar epithelial permeability following mechanical ventilation leads to increased BALF p75 and p55 via plasma leakage. These data provide new insights into differential regulation of intra-alveolar sTNFR levels during ALI and may suggest sTNFRs as potential markers for evaluating the pathophysiology of ALI.

Inhibition of TNF Receptor p55 By a Domain Antibody Attenuates the Initial Phase of Acid-Induced Lung Injury in Mice

Background Tumor necrosis factor-α (TNF) is strongly implicated in the development of acute respiratory distress syndrome (ARDS), but its potential as a therapeutic target has been hampered by its complex biology. TNF signals through two receptors, p55 and p75, which play differential roles in pulmonary edema formation during ARDS. We have recently shown that inhibition of p55 by a novel domain antibody (dAb™) attenuated ventilator-induced lung injury. In the current study, we explored the efficacy of this antibody in mouse models of acid-induced lung injury to investigate the longer consequences of treatment. Methods We employed two acid-induced injury models, an acute ventilated model and a resolving spontaneously breathing model. C57BL/6 mice were pretreated intratracheally or intranasally with p55-targeting dAb or non-targeting “dummy” dAb, 1 or 4 h before acid instillation. Results Acid instillation in the dummy dAb group caused hypoxemia, increased respiratory system elastance, pulmonary inflammation, and edema in both the ventilated and resolving models. Pretreatment with p55-targeting dAb significantly attenuated physiological markers of ARDS in both models. p55-targeting dAb also attenuated pulmonary inflammation in the ventilated model, with signs that altered cytokine production and leukocyte recruitment persisted beyond the very acute phase. Conclusion These results demonstrate that the p55-targeting dAb attenuates lung injury and edema formation in models of ARDS induced by acid aspiration, with protection from a single dose lasting up to 24 h. Together with our previous data, the current study lends support toward the clinical targeting of p55 for patients with, or at risk of ARDS.

Intravascular donor monocytes play a central role in lung transplant ischaemia-reperfusion injury

Rationale Primary graft dysfunction in lung transplant recipients derives from the initial, largely leukocyte-dependent, ischaemia-reperfusion injury. Intravascular lung-marginated monocytes have been shown to play key roles in experimental acute lung injury, but their contribution to lung ischaemia-reperfusion injury post transplantation is unknown. Objective To define the role of donor intravascular monocytes in lung transplant-related acute lung injury and primary graft dysfunction. Methods Isolated perfused C57BL/6 murine lungs were subjected to warm ischaemia (2 hours) and reperfusion (2 hours) under normoxic conditions. Monocyte retention, activation phenotype and the effects of their depletion by intravenous clodronate-liposome treatment on lung inflammation and injury were determined. In human donor lung transplant samples, the presence and activation phenotype of monocytic cells (low side scatter, 27E10+, CD14+, HLA-DR+, CCR2+) were evaluated by flow cytometry and compared with post-implantation lung function. Results In mouse lungs following ischaemia-reperfusion, substantial numbers of lung-marginated monocytes remained within the pulmonary microvasculature, with reduced L-selectin and increased CD86 expression indicating their activation. Monocyte depletion resulted in reductions in lung wet:dry ratios, bronchoalveolar lavage fluid protein, and perfusate levels of RAGE, MIP-2 and KC, while monocyte repletion resulted in a partial restoration of the injury. In human lungs, correlations were observed between pre-implantation donor monocyte numbers/their CD86 and TREM-1 expression and post-implantation lung dysfunction at 48 and 72 hours. Conclusions These results indicate that lung-marginated intravascular monocytes are retained as a ‘passenger’ leukocyte population during lung transplantation, and play a key role in the development of transplant-associated ischaemia-reperfusion injury.

105: MULTIDISCIPLINARY ROUNDS SHORTEN ICU LENGTH OF STAY IN A JAPANESE UNIVERSITY TERTIARY HOSPITAL

Learning Objectives: Care of critically ill patients is complex and requires input from various experts. However, coordinating a platform to discuss inputs in a multidisciplinary fashion is challenging due to conflicts in schedule and interests. Prior studies in the US revealed hospitals that conducted multidisciplinary rounds improved outcomes in the ICU. In Japan, although the concept of ICU multidisciplinary rounds has gained interest, it is still in the infant stages. We sought to report for the first time the effect of ICU multidisciplinary rounds at a university tertiary hospital in Japan. Methods: We conducted a retrospective observational study of patients who were admitted to our semi-closed ICU. We compared the average ICU length of stay 6 months before and after the implementation of multidisciplinary rounds (pre: between October 2015 and March 2016, post: between October 2016 and March 2017) consisting of ICU nurse, pharmacist, nutritionist, physical therapist, medical engineers, and infection control nurse. We also compared non-cardiac patients and cardiac patients as subgroup analysis. The mean central venous (CV) catheter days was compared as a secondary outcome. Results: A total of 769 patients were included. There was no significant difference in patient characteristics. The average ICU length of stay significantly decreased by 12% (5.5 ± 9.2 days vs. 5.0 ± 6.1 days, p = 0.029). Furthermore, the average ICU length of stay of non-cardiac patients significantly decreased by 23% (4.6 ± 7.1 days vs. 3.8 ± 3.9 days, p < 0.001), whereas that of cardiac patients were not significantly different (8.3 ± 13.2 days vs. 8.1 ± 9.1 days). The mean CV catheter days showed a reduction of 41% after intervention (7.0 ± 1.4 days vs. 5.0 ± 0.6 days, p = 0.007). Conclusions: Multidisciplinary rounds significantly decreased ICU length of stay and mean CV catheter days in Japanese university tertiary hospital. Although there may be differences in practice and structure of ICU care compared to the US, our findings suggest that the systemic implementation of ICU multidisciplinary rounds improves the ICU outcomes.

The role of ex vivo lung perfusion in lung transplantation

Whilst lung transplantation is a viable solution for end-stage lung disease, donor shortages, donor lung inflammation and perioperative lung injury remain major limitations. Ex vivo lung perfusion has emerged as the next frontier in lung transplantation to address and overcome these limitations, with multicentre clinical trials ongoing in the UK, rest of Europe and North America. Our research seeks to identify the poorly understood cellular and molecular mechanisms of primary graft dysfunction through the development of an isolated perfused lung model of transplantation and investigation of the role of pulmonary inflammation in this paradigm.

S107 Comparison of high-stretch versus atelectasis in the pathophysiology of ventilator-induced lung injury using the mouse isolated perfused lung

Introduction Mechanical ventilation promotes pulmonary inflammation and oedema formation, a process known as ventilator-induced lung injury (VILI). Various aspects of ventilation have been proposed as injurious, including over distension of alveoli and the repetitive collapse and reopening of lung units associated with atelectasis. Here we attempt to elucidate the impact of these distinct entities on the pathophysiology of VILI, using a mouse isolated perfused lung, which enables us to study the effects of stretch versus atelectasis in the absence of extra pulmonary factors. Methods Lungs were obtained from male C57BL6 mice, and allocated to one of three groups, that is, control, atelectasis or high-stretch. All the lungs were ventilated with respiratory rate of 80/min, and perfused at 25 ml/kg/min in a recirculating manner with non-blood buffer for 3 h. In the control group, low tidal volume (7 ml/kg) with positive end-expiratory pressure (PEEP; 5 cmH2O) and regular deep inflation (DI; 25 cmH2O, every 15 min) was applied. The atelectasis group received the same low tidal volume, but neither PEEP nor DI. In the high-stretch group, lungs were ventilated with high tidal volume (30–32 ml/kg) and both PEEP (3 cmH2O) and DI. Perfusate and lung lavage samples were taken at the end of experiments for analysis of total protein and chemokines. Results The lungs in the atelectasis and high-stretch groups developed similar, severe pulmonary oedema as represented by increases in protein levels in lavage fluid. High-stretch induced substantial increases in both perfusate and lavage fluid chemokines, compared to controls. In stark contrast, the atelectasis group showed similar low levels of chemokines in perfusate, with only slight increases in lavage fluid chemokines, compared to controls (Abstract S107 Table 1).Abstract S107 Table 1 Control (n=5) High-stretch (n=3) Atelectasis (n=5) Protein in lavage fluid (mg/ml) 0.21±0.04 4.07±2.21* 4.81±1.67* Perfusate chemokines  MIP-2 (ng/ml) 1.12±0.70 4.29±0.82* † 1.48±0.67  KC (ng/ml) 1.26±0.74 6.71±1.79* † 1.70±1.09 Lavage fluid chemokines  MIP-2 (ng/ml) 0.13±0.06 4.39±1.98* † 0.69±0.26*  KC (ng/ml) 0.12±0.04 5.15±1.42* † 0.82±0.47** p<0.05 versus control.† p<0.05 versus atelectasis. MIP-2, Macrophage-inflammatory protein-2; KC, Kerationocyte-derived chemokine. Conclusion While both high-stretch and atelectasis can induce barrier dysfunction, only high-stretch induced substantial production of chemokines by the lung and their release into the circulation. These findings suggest that over distension of the lung, rather than collapse/reopening associated with atelectasis, primarily contributes to the exacerbated pulmonary and systemic inflammation during VILI. Our results may provide insights into why addition of PEEP to limit atelectasis has limited clinical outcome benefit in ventilated patients with acute lung injury.