Maria T. Kuipers

Ventilator-induced lung injury is mediated by the NLRP3 inflammasome

Background: The innate immune response is important in ventilator-induced lung injury (VILI) but the exact pathways involved are not elucidated. The authors studied the role of the intracellular danger sensor NLRP3 inflammasome. Methods: NLRP3 inflammasome gene expression was analyzed in respiratory epithelial cells and alveolar macrophages obtained from ventilated patients (n = 40). In addition, wild-type and NLRP3 inflammasome deficient mice were randomized to low tidal volume (approximately 7.5 ml/kg) and high tidal volume (approximately 15 ml/kg) ventilation. The presence of uric acid in lung lavage, activation of caspase-1, and NLRP3 inflammasome gene expression in lung tissue were investigated. Moreover, mice were pretreated with interleukin-1 receptor antagonist, glibenclamide, or vehicle before start of mechanical ventilation. VILI endpoints were relative lung weights, total protein in lavage fluid, neutrophil influx, and pulmonary and systemic cytokine and chemokine concentrations. Data represent mean ± SD. Results: Mechanical ventilation up-regulated messenger RNA expression levels of NLRP3 in alveolar macrophages (1.0 ± 0 vs. 1.70 ± 1.65, P less than 0.05). In mice, mechanical ventilation increased both NLRP3 and apoptosis-associated speck-like protein messenger RNA levels, respectively (1.08 ± 0.55 vs. 3.98 ± 2.89; P less than 0.001 and 0.95 ± 0.53 vs. 6.0 ± 3.55; P less than 0.001), activated caspase-1, and increased uric acid levels (6.36 ± 1.85 vs. 41.9 ± 32.0, P less than 0.001). NLRP3 inflammasome deficient mice displayed less VILI due to high tidal volume mechanical ventilation compared with wild-type mice. Furthermore, treatment with interleukin-1 receptor antagonist or glibenclamide reduced VILI. Conclusions: Mechanical ventilation induced a NLRP3 inflammasome dependent pulmonary inflammatory response. NLRP3 inflammasome deficiency partially protected mice from VILI.

Bench-to-bedside review: Damage-associated molecular patterns in the onset of ventilator- induced lung injury

Mechanical ventilation (MV) has the potential to worsen pre-existing lung injury or even to initiate lung injury. Moreover, it is thought that injurious MV contributes to the overwhelming inflammatory response seen in patients with acute lung injury or acute respiratory distress syndrome. Ventilator-induced lung injury (VILI) is characterized by increased endothelial and epithelial permeability and pulmonary inflammation, in which the innate immune system plays a key role. A growing body of evidence indicates that endogenous danger molecules, also termed damage-associated molecular patterns (DAMPs), are released upon tissue injury and modulate the inflammatory response. DAMPs activate pattern recognition receptors, may induce the release of proinflammatory cytokines and chemokines, and have been shown to initiate or propagate inflammation in non-infectious conditions. Experimental and clinical studies demonstrate the presence of DAMPs in bronchoalveolar lavage fluid in patients with VILI and the upregulation of pattern recognition receptors in lung tissue by MV. The objective of the present article is to review research in the area of DAMPs, their recognition by the innate immune system, their role in VILI, and the potential utility of blocking DAMP signaling pathways to reduce VILI in the critically ill.

Hydrogen sulfide donor NaHS reduces organ injury in a rat model of pneumococcal pneumosepsis, associated with improved bio-energetic status.

Sepsis is characterized by a generalized inflammatory response and organ failure, associated with mitochondrial dysfunction. Hydrogen sulfide donor NaHS has anti-inflammatory properties, is able to reduce metabolism and can preserve mitochondrial morphology and function. Rats were challenged with live Streptococcus pneumonia or saline and infused with NaHS (36 µmol/kg/h) or vehicle. Lung and kidney injury markers were measured as well as mitochondrial function, viability and biogenesis. Infusion of NaHS reduced heart rate and body temperature, indicative of a hypo–metabolic state. NaHS infusion reduced sepsis–related lung and kidney injury, while host defense remained intact, as reflected by unchanged bacterial outgrowth. The reduction in organ injury was associated with a reversal of a fall in active oxidative phosphorylation with a concomitant decrease in ATP levels and ATP/ADP ratio. Preservation of mitochondrial respiration was associated with increased mitochondrial expression of α–tubulin and protein kinase C–ε, which acts as regulators of respiration. Mitochondrial damage was decreased by NaHS, as suggested by a reduction in mitochondrial DNA leakage in the lung. Also, NaHS treatment was associated with upregulation of peroxisome proliferator-activated receptor–γ coactivator 1α, with a subsequent increase in transcription of mitochondrial respiratory subunits. These findings indicate that NaHS reduces organ injury in pneumosepsis, possibly via preservation of oxidative phosphorylation and thereby ATP synthesis as well as by promoting mitochondrial biogenesis. Further studies on the involvement of mitochondria in sepsis are required.

A short course of infusion of a hydrogen sulfide-donor attenuates endotoxemia induced organ injury via stimulation of anti-inflammatory pathways, with no additional protection from prolonged infusion.

Organ failure is associated with increased mortality and morbidity in patients with systemic inflammatory response syndrome. Previously, we showed that a short course of infusion of a hydrogen sulfide (H(2)S) donor reduced metabolism with concurrent reduction of lung injury. Here, we hypothesize that prolonged H(2)S infusion is more protective than a short course in endotoxemia with organ failure. Also, as H(2)S has both pro- and anti-inflammatory effects, we explored the effect of H(2)S on interleukin production. Endotoxemia was induced by an intravenous bolus injection of LPS (7.5mg/kg) in mechanically ventilated rats. H(2)S donor NaHS (2mg/kg) or vehicle (saline) was infused and organ injury was determined after either 4 or 8h. A short course of H(2)S infusion was associated with reduction of lung and kidney injury. Prolonged infusion did not enhance protection. Systemically, infusion of H(2)S increased both the pro-inflammatory response during endotoxemia, as demonstrated by increased TNF-α levels, as well as the anti-inflammatory response, as demonstrated by increased IL-10 levels. In LPS-stimulated whole blood of healthy volunteers, co-incubation with H(2)S had solely anti-inflammatory effects, resulting in decreased TNF-α levels and increased IL-10 levels. Co-incubation with a neutralizing IL-10 antibody partly abrogated the decrease in TNF-α levels. In conclusion, a short course of H(2)S infusion reduced organ injury during endotoxemia, at least in part via upregulation of IL-10.

High Levels of S100A8/A9 Proteins Aggravate Ventilator-Induced Lung Injury via TLR4 Signaling

Background Bacterial products add to mechanical ventilation in enhancing lung injury. The role of endogenous triggers of innate immunity herein is less well understood. S100A8/A9 proteins are released by phagocytes during inflammation. The present study investigates the role of S100A8/A9 proteins in ventilator-induced lung injury. Methods Pulmonary S100A8/A9 levels were measured in samples obtained from patients with and without lung injury. Furthermore, wild-type and S100A9 knock-out mice, naive and with lipopolysaccharide-induced injured lungs, were randomized to 5 hours of spontaneously breathing or mechanical ventilation with low or high tidal volume (VT). In addition, healthy spontaneously breathing and high VT ventilated mice received S100A8/A9, S100A8 or vehicle intratracheal. Furthermore, the role of Toll-like receptor 4 herein was investigated. Results S100A8/A9 protein levels were elevated in patients and mice with lung injury. S100A8/A9 levels synergistically increased upon the lipopolysaccharide/high VT MV double hit. Markers of alveolar barrier dysfunction, cytokine and chemokine levels, and histology scores were attenuated in S100A9 knockout mice undergoing the double-hit. Exogenous S100A8/A9 and S100A8 induced neutrophil influx in spontaneously breathing mice. In ventilated mice, these proteins clearly amplified inflammation: neutrophil influx, cytokine, and chemokine levels were increased compared to ventilated vehicle-treated mice. In contrast, administration of S100A8/A9 to ventilated Toll-like receptor 4 mutant mice did not augment inflammation. Conclusion S100A8/A9 proteins increase during lung injury and contribute to inflammation induced by HVT MV combined with lipopolysaccharide. In the absence of lipopolysaccharide, high levels of extracellular S100A8/A9 still amplify ventilator-induced lung injury via Toll-like receptor 4.

Mechanical Ventilation and the Titer of Antibodies as Risk Factors for the Development of Transfusion-Related Lung Injury

Purpose. Onset of transfusion-related acute lung injury (TRALI) is suggested to be a threshold-event. Data is lacking on the relation between titer of antibodies infused and onset of TRALI. We determined whether onset of TRALI is dependent on the titer of MHC-I antibodies infused in a combined model of ventilator-induced lung injury and antibody-induced TRALl. Methods. BALB/c mice were ventilated for five hours with low (7.5 ml/kg) or high (15 ml/kg) tidal volume. After three hours of MV, TRALI was induced by infusion of 0.5 mg/kg, 2.0 mg/kg or 4.5 mg/kg MHC-I antibodies. Control animals received vehicle. After five hours of MV, animals were sacrificed. Results. MV with high tidal volumes resulted in increased levels of all markers of lung injury compared to animals ventilated with low tidal MV. In ventilator-induced lung injury, infusion of 4.5 mg/kg of antibodies further increased pulmonary wet-to-dry ratio, pulmonary neutrophil influx and pulmonary KC levels, whereas infusion of lower dose of antibodies did not augment lung injury. In contrast, mice ventilated with low tidal volumes did not develop lung injury, irrespective of the dose of antibody used. Conclusions. In the presence of injurious MV, onset of TRALI depends on the titer of antibodies infused.

The caspase inhibitor zVAD increases lung inflammation in pneumovirus infection in mice

Severe respiratory syncytial virus (RSV) disease is a frequent cause of acute respiratory distress syndrome (ARDS) in young children, and is associated with marked lung epithelial injury and neutrophilic inflammation. Experimental studies on ARDS have shown that inhibition of apoptosis in the lungs reduces lung epithelial injury. However, the blockade of apoptosis in the lungs may also have deleterious effects by hampering viral clearance, and importantly, by enhancing or prolonging local proinflammatory responses. The aim of this study was to determine the effect of the broad caspase inhibitor Z‐VAD(OMe)‐FMK (zVAD) on inflammation and lung injury in a mouse pneumovirus model for severe RSV disease. Eight‐ to 11‐week‐old female C57BL/6OlaHsd mice were inoculated with the rodent‐specific pneumovirus pneumonia virus of mice (PVM) strain J3666 and received multiple injections of zVAD or vehicle (control) during the course of disease, after which they were studied for markers of apoptosis, inflammation, and lung injury on day 7 after infection. PVM‐infected mice that received zVAD had a strong increase in neutrophil numbers in the lungs, which was associated with decreased neutrophil apoptosis. Furthermore, zVAD treatment led to higher concentrations of several proinflammatory cytokines in the lungs and more weight loss in PVM‐infected mice. In contrast, zVAD did not reduce apoptosis of lung epithelial cells and did not affect the degree of lung injury, permeability, and viral titers in PVM disease. We conclude that zVAD has an adverse effect in severe pneumovirus disease in mice by enhancing the lung proinflammatory response.

Induced hypothermia is protective in a rat model of pneumococcal pneumonia.

Induced hypothermia is protective in ischemia-reperfusion injury by reducing the inflammatory response and is increasingly applied in the ICU. Hypothermia may dampen host response during an infection and it is believed that induced hypothermia may carry the risk of acquiring or aggravating an infection. We investigated the effect of hypothermia on bacterial outgrowth and on the inflammatory response in a rat model of pneumococcal pneumonia.