TLR2 deficiency aggravates lung injury caused by mechanical ventilation

Innate immunity pathways are found to play an important role in ventilator-induced lung injury. We analyzed pulmonary expression of Toll-like receptor 2 (TLR2) in humans and mice and determined the role of TLR2 in the pathogenesis of ventilator-induced lung injury in mice. Toll-like receptor 2 gene expression was analyzed in human bronchoalveolar lavage fluid (BALF) cells and murine lung tissue after 5 h of ventilation. In addition, wild-type (WT) and TLR2 knockout (KO) mice were ventilated with either lower tidal volumes (VT) of 7 mL/kg with positive end-expiratory pressure (PEEP) or higher VT of 15 mL/kg without PEEP for 5 h. Spontaneously breathing mice served as controls. Total protein and immunoglobulin M levels in BALF, neutrophil influx into the alveolar compartment, and interleukin 6 (IL-6), IL-1β, and keratinocyte-derived chemokine concentrations in lung tissue homogenates were measured. We observed enhanced TLR2 gene expression in BALF cells of ventilated patients and in lung tissue of ventilated mice. In WT mice, ventilation with higher VT without PEEP resulted in lung injury and inflammation with higher immunoglobulin M levels, neutrophil influx, and levels of inflammatory mediators compared with controls. In TLR2 KO mice, neutrophil influx and IL-6, IL-1β, and keratinocyte-derived chemokine were enhanced by this ventilation strategy. Ventilation with lower VT with PEEP only increased neutrophil influx and was similar in WT and TLR2 KO mice. In summary, injurious ventilation enhances TLR2 expression in lungs. Toll-like receptor 2 deficiency does not protect lungs from ventilator-induced lung injury. In contrast, ventilation with higher VT without PEEP aggravates inflammation in TLR2 KO mic

Prolonged Helium Postconditioning Protocols during Early Reperfusion Do Not Induce Cardioprotection in the Rat Heart In Vivo: Role of Inflammatory Cytokines

Postconditioning of myocardial tissue employs short cycles of ischemia or pharmacologic agents during early reperfusion. Effects of helium postconditioning protocols on infarct size and the ischemia/reperfusion-induced immune response were investigated by measurement of protein and mRNA levels of proinflammatory cytokines. Rats were anesthetized with S-ketamine (150 mg/kg) and diazepam (1.5 mg/kg). Regional myocardial ischemia/reperfusion was induced; additional groups inhaled 15, 30, or 60 min of 70% helium during reperfusion. Fifteen minutes of helium reduced infarct size from 43% in control to 21%, whereas 30 and 60 minutes of helium inhalation led to an infarct size of 47% and 39%, respectively. Increased protein levels of cytokine-induced neutrophil chemoattractant (CINC-3) and interleukin-1 beta (IL-1 ) were found after 30 or 60 min of helium inhalation, in comparison to control. 30 min of helium increased mRNA levels of CINC-3, IL-1 , interleukin 6 (IL-6), and tumor necrosis factor alpha (TNF-) in myocardial tissue not directly subjected to ischemia/reperfusion. These results suggest that the effectiveness of the helium postconditioning protocol is very sensitive to duration of noble gas application. Additionally, helium was associated with higher levels of inflammatory cytokines; however, it is not clear whether this is causative of nature or part of an epiphenomenon

Prolonged Helium Postconditioning Protocols during Early Reperfusion Do Not Induce Cardioprotection in the Rat Heart In Vivo: Role of Inflammatory Cytokines

Postconditioning of myocardial tissue employs short cycles of ischemia or pharmacologic agents during early reperfusion. Effects of helium postconditioning protocols on infarct size and the ischemia/reperfusion-induced immune response were investigated by measurement of protein and mRNA levels of proinflammatory cytokines. Rats were anesthetized with S-ketamine (150 mg/kg) and diazepam (1.5 mg/kg). Regional myocardial ischemia/reperfusion was induced; additional groups inhaled 15, 30, or 60 min of 70% helium during reperfusion. Fifteen minutes of helium reduced infarct size from 43% in control to 21%, whereas 30 and 60 minutes of helium inhalation led to an infarct size of 47% and 39%, respectively. Increased protein levels of cytokine-induced neutrophil chemoattractant (CINC-3) and interleukin-1 beta (IL-1β) were found after 30 or 60 min of helium inhalation, in comparison to control. 30 min of helium increased mRNA levels of CINC-3, IL-1β, interleukin 6 (IL-6), and tumor necrosis factor alpha (TNF-α) in myocardial tissue not directly subjected to ischemia/reperfusion. These results suggest that the effectiveness of the helium postconditioning protocol is very sensitive to duration of noble gas application. Additionally, helium was associated with higher levels of inflammatory cytokines; however, it is not clear whether this is causative of nature or part of an epiphenomenon

Pre-Treatment with Allopurinol or Uricase Attenuates Barrier Dysfunction but Not Inflammation during Murine Ventilator-Induced Lung Injury

<div><h3>Introduction</h3><p>Uric acid released from injured tissue is considered a major endogenous danger signal and local instillation of uric acid crystals induces acute lung inflammation via activation of the NLRP3 inflammasome. Ventilator-induced lung injury (VILI) is mediated by the NLRP3 inflammasome and increased uric acid levels in lung lavage fluid are reported. We studied levels in human lung injury and the contribution of uric acid in experimental VILI.</p> <h3>Methods</h3><p>Uric acid levels in lung lavage fluid of patients with acute lung injury (ALI) were determined. In a different cohort of cardiac surgery patients, uric acid levels were correlated with pulmonary leakage index. In a mouse model of VILI the effect of allopurinol (inhibits uric acid synthesis) and uricase (degrades uric acid) pre-treatment on neutrophil influx, up-regulation of adhesion molecules, pulmonary and systemic cytokine levels, lung pathology, and regulation of receptors involved in the recognition of uric acid was studied. In addition, total protein and immunoglobulin M in lung lavage fluid and pulmonary wet/dry ratios were measured as markers of alveolar barrier dysfunction.</p> <h3>Results</h3><p>Uric acid levels increased in ALI patients. In cardiac surgery patients, elevated levels correlated significantly with the pulmonary leakage index. Allopurinol or uricase treatment did not reduce ventilator-induced inflammation, IκB-α degradation, or up-regulation of NLRP3, Toll-like receptor 2, and Toll-like receptor 4 gene expression in mice. Alveolar barrier dysfunction was attenuated which was most pronounced in mice pre-treated with allopurinol: both treatment strategies reduced wet/dry ratio, allopurinol also lowered total protein and immunoglobulin M levels.</p> <h3>Conclusions</h3><p>Local uric acid levels increase in patients with ALI. In mice, allopurinol and uricase attenuate ventilator-induced alveolar barrier dysfunction.</p> </div

The receptor for advanced glycation end products in ventilator-induced lung injury

Mechanical ventilation (MV) can cause ventilator-induced lung injury (VILI). The innate immune response mediates this iatrogenic inflammatory condition. The receptor for advanced glycation end products (RAGE) is a multiligand receptor that can amplify immune and inflammatory responses. We hypothesized that RAGE signaling contributes to the pro-inflammatory state induced by MV. RAGE expression was analyzed in lung brush and lavage cells obtained from ventilated patients and lung tissue of ventilated mice. Healthy wild-type (WT) and RAGE knockout (KO) mice were ventilated with relatively low (approximately 7.5 ml/kg) or high (approximately 15 ml/kg) tidal volume. Positive end-expiratory pressure was set at 2 cm H2O during both MV strategies. Also, WT and RAGE KO mice with lipopolysaccharide (LPS)-induced lung injury were ventilated with the above described ventilation strategies. In separate experiments, the contribution of soluble RAGE, a RAGE isoform that may function as a decoy receptor, in ventilated RAGE KO mice was investigated. Lung wet-to-dry ratio, cell and neutrophil influx, cytokine and chemokine concentrations, total protein levels, soluble RAGE, and high-mobility group box 1 (HMGB1) presence in lung lavage fluid were analyzed. MV was associated with increased RAGE mRNA levels in both human lung brush samples and lung tissue of healthy mice. In healthy high tidal volume-ventilated mice, RAGE deficiency limited inflammatory cell influx. Other VILI parameters were not affected. In our second set of experiments where we compared RAGE KO and WT mice in a 2-hit model, we observed higher pulmonary cytokine and chemokine levels in RAGE KO mice undergoing LPS/high tidal volume MV as compared to WT mice. Third, in WT mice undergoing the LPS/high tidal volume MV, we observed HMGB1 presence in lung lavage fluid. Moreover, MV increased levels of soluble RAGE in lung lavage fluid, with the highest levels found in LPS/high tidal volume-ventilated mice. Administration of soluble RAGE to LPS/high tidal volume-ventilated RAGE KO mice attenuated the production of inflammatory mediators. RAGE was not a crucial contributor to the pro-inflammatory state induced by MV. However, the presence of sRAGE limited the production of pro-inflammatory mediators in our 2-hit model of LPS and high tidal volume M

Pulmonary mRNA levels of pattern-recognition receptors.

<p>Animals were pre-treated with uricase (0.2 mg/kg), allopurinol (25 mg/kg), or vehicle (10% dimethylsulfoxide) and were mechanically ventilated for 5 hours (MV), non-ventilated vehicle pre-treated mice served as controls (C). Gene expression levels of Toll-like receptor (TLR) 2, TLR4, and, NLRP3 were measured in lung tissue homogenates and normalized to the house-keeping gene hypoxanthine-guaninephosphoribosyl transferase (HPRT). Data represent mean ± SEM of n = 4 non-ventilated mice and n = 8−9 ventilated mice/group.</p>**<p>p<0.01 versus non-ventilated controls.</p

Histopathology of VILI in mice is affected by allopurinol pre-treatment.

<p>Lung tissue slides of mice pre-treated with vehicle (10% dimethylsulfoxide, dark grey bars), uricase (0.2 mg/kg, light grey, striped bars), or allopurinol (25 mg/kg (white bars) 1 hour before start of mechanical ventilation (VILI). Spontaneously breathing, vehicle pre-treated mice served as controls (C). Lungs were scored for presence of edema, hemorrhage, neutrophil influx, and hyaline membranes. Total scores and edema scores are demonstrated below. Data are presented as mean ± SEM of 4 control mice and n = 9 for the VILI groups. *p<0.05 vs vehicle control H&E staining, magnification ×20.</p

Uric acid levels in murine ventilator-induced lung injury.

<p>Uric acid levels in plasma (<b>A</b>) and bronchoalveolar lavage fluid (BALF) (<b>B</b>) of mice pre-treated with vehicle (10% dimethylsulfoxide, dark grey bars), uricase (0.2 mg/kg, light grey, striped bars), or allopurinol (25 mg/kg (white bars) 1 hour before start of 5 hours of mechanical ventilation (VILI). Spontaneously breathing, vehicle pre-treated mice served as controls (C). Data represent mean ± SEM of 4 control mice and n = 6−9 ventilated mice. **p<0.01 vs. control, #p<0.05 vs. vehicle ventilated.</p

Allopurinol and uricase pre-treatment attenuate alveolar barrier dysfunction.

<p>Mice were pre-treated with vehicle (10% dimethylsulfoxide, dark grey bars), uricase (0.2 mg/kg, light grey, striped bars), or allopurinol (25 mg/kg (white bars) 1 hour before start of mechanical ventilation (VILI). Spontaneously breathing, vehicle pre-treated mice served as controls (C). After 5 hours mice were killed. Lung wet-to-dry ratio (<b>A</b>), total protein levels in lung lavage fluid (<b>B</b>) and immunoglobulin M (IgM) concentrations in (<b>C</b>) were analyzed. Data represent mean ± SEM of 4 control mice and n = 9 for the VILI groups (for IgM n = 5−6). ##P<0.01, ###P<0.001 vs. vehicle ventilated. ***p<0.001 vs. vehicle control.</p

Hemodynamic parameters during 5 hours of ventilation in mice.

<p>Hemodynamic parameters of mice pre-treated with vehicle (10% dimethylsulfoxide), uricase (0.2 mg/kg), or allopurinol (25 mg/kg) 1 hour before start of mechanical ventilation. Systolic bloodpressure (<b>A</b>) and heart rate (<b>B</b>) was measured at start of ventilation, after 2.5, and after 5 hours. Data represent mean ± SEM of n = 9 mice per group.</p