Heat-shock response increases lung injury caused by Pseudomonas aeruginosa via an interleukin-10-dependent mechanism in mice.

BACKGROUND: The heat-shock response (HSR) protects from insults, such as ischemia-reperfusion injury, by inhibiting signaling pathways activated by sterile inflammation. However, the mechanisms by which the HSR activation would modulate lung damage and host response to a bacterial lung infection remain unknown. METHODS: HSR was activated with whole-body hyperthermia or by intraperitoneal geldanamycin in mice that had their lungs instilled with Pseudomonas aeruginosa 24 h later (at least six mice per experimental group). Four hours after instillation, lung endothelial and epithelial permeability, bacterial counts, protein levels in bronchoalveolar lavage fluid, and lung myeloperoxidase activity were measured. Mortality rate 24 h after P. aeruginosa instillation was recorded. The HSR effect on the release of interleukin-10 and killing of P. aeruginosa bacteria by a mouse alveolar macrophage cell line and on neutrophil phagocytosis was also examined. RESULTS: HSR activation worsened lung endothelial (42%) and epithelial permeability (50%) to protein, decreased lung bacterial clearance (71%), and increased mortality (50%) associated with P. aeruginosa pneumonia, an effect that was not observed in heat-shock protein-72-null mice. HSR-mediated decrease in neutrophil phagocytosis (69%) and bacterial killing (38%) by macrophages was interleukin-10 dependent, a mechanism confirmed by increased lung bacterial clearance and decreased mortality (70%) caused by P. aeruginosa pneumonia in heat-shocked interleukin-10-null mice. CONCLUSIONS: Prior HSR activation worsens lung injury associated with P. aeruginosa pneumonia in mice via heat-shock protein-72- and interleukin-10-dependent mechanisms. These results provide a novel mechanism for the immunosuppression observed after severe trauma that is known to activate HSR in humans.

Stroke-induced activation of the α7 nicotinic receptor increases Pseudomonas aeruginosa lung injury.

Infectious complications, predominantly pneumonia, are the most common cause of death in the postacute phase of stroke, although the mechanisms underlying the corresponding immunosuppression are not fully understood. We tested the hypothesis that activation of the α7 nicotinic acetylcholine receptor (α7nAChR) pathway is important in the stroke-induced increase in lung injury caused by Pseudomonas aeruginosa pneumonia in mice. Prior stroke increased lung vascular permeability caused by P. aeruginosa pneumonia and was associated with decreased lung neutrophil recruitment and bacterial clearance in mice. Pharmacologic inhibition (methyllycaconitine IC(50): 0.2-0.6 nM) or genetic deletion of the α7nAChR significantly (P<0.05) attenuates the effect of prior stroke on lung injury and mortality caused by P. aeruginosa pneumonia in mice. Finally, pretreatment with PNU-282987, a pharmacologic activator of the α7nAChR (EC(50): 0.2 µM), significantly (P<0.05) increased lung injury caused by P. aeruginosa pneumonia, significantly (P<0.05) decreased the release of KC, a major neutrophil chemokine, and significantly (P<0.05) decreased intracellular bacterial killing by a mouse alveolar macrophage cell line and primary mouse neutrophils. In summary, the α7 nicotinic cholinergic pathway plays an important role in mediating the systemic immunosuppression observed after stroke and directly contributes to more severe lung damage induced by P. aeruginosa.

PAI-1 is an essential component of the pulmonary host response during Pseudomonas aeruginosa pneumonia in mice.

RATIONALE: Elevated plasma and bronchoalveolar lavage fluid plasminogen activator inhibitor 1 (PAI-1) levels are associated with adverse clinical outcome in patients with pneumonia caused by Pseudomonas aeruginosa. However, whether PAI-1 plays a pathogenic role in the breakdown of the alveolar-capillary barrier caused by P aeruginosa is unknown. OBJECTIVES: The role of PAI-1 in pulmonary host defence and survival during P aeruginosa pneumonia in mice was tested. The in vitro mechanisms by which P aeruginosa causes PAI-1 gene and protein expression in lung endothelial and epithelial cells were also examined. METHODS AND RESULTS: PAI-1 null and wild-type mice that were pretreated with the PAI-1 inhibitor Tiplaxtinin had a significantly lower increase in lung vascular permeability than wild-type littermates after the airspace instillation of 1×10(7) colony-forming units (CFU) of P aeruginosa bacteria. Furthermore, P aeruginosa in vitro induced the expression of the PAI-1 gene and protein in a TLR4/p38/RhoA/NF-κB (Toll-like receptor 4/p38/RhoA/nuclear factor-κB) manner in lung endothelial and alveolar epithelial cells. However, in vivo disruption of PAI-1 signalling was associated with higher mortality at 24 h (p<0.03) and higher bacterial burden in the lungs secondary to decreased neutrophil migration into the distal airspace in response to P aeruginosa. CONCLUSIONS: The results indicate that PAI-1 is a critical mediator that controls the development of the early lung inflammation that is required for the activation of the later innate immune response necessary for the eradication of P aeruginosa from the distal airspaces of the lung.

Cytoprotective-selective activated protein C attenuates Pseudomonas aeruginosa-induced lung injury in mice.

Inhibition of the small GTPase RhoA attenuates the development of pulmonary edema and restores positive alveolar fluid clearance in a murine model of Pseudomonas aeruginosa pneumonia. Activated protein C (aPC) blocks the development of an unfavorably low ratio of small GTPase Rac1/RhoA activity in lung endothelium through endothelial protein C receptor (EPCR)/protease-activated receptor-1 (PAR-1)-dependent signaling mechanisms that include transactivating the sphingosine-1-phosphate (S1P) pathway. However, whether aPC's cytoprotective effects can attenuate the development of pulmonary edema and death associated with P. aeruginosa pneumonia in mice remains unknown. Thus, we determined whether the normalization of a depressed ratio of activated Rac1/RhoA by aPC would attenuate the P. aeruginosa-mediated increase in protein permeability across lung endothelial and alveolar epithelial barriers. Pretreatment with aPC significantly reduced P. aeruginosa-induced increases in paracellular permeability across pulmonary endothelial cell and alveolar epithelial monolayers via an inhibition of RhoA activation and a promotion of Rac1 activation that required the EPCR-PAR-1 and S1P pathways. Furthermore, pretreatment with aPC attenuated the development of pulmonary edema in a murine model of P. aeruginosa pneumonia. Finally, a cytoprotective-selective aPC mutant, aPC-5A, which lacks most of aPC's anticoagulant activity, reproduced the protective effect of wild-type aPC by attenuating the development of pulmonary edema and decreasing mortality in a murine model of P. aeruginosa pneumonia. Taken together, these results demonstrate a critical role for the cytoprotective activities of aPC in attenuating P. aeruginosa-induced lung vascular permeability and mortality, suggesting that cytoprotective-selective aPC-5A with diminished bleeding risks could attenuate the lung damage caused by P. aeruginosa in critically ill patients.

Critical role of the small GTPase RhoA in the development of pulmonary edema induced by Pseudomonas aeruginosa in mice.

BACKGROUND: Pseudomonas aeruginosa is an opportunistic pathogen that can cause severe pneumonia in critically ill patients. We have reported previously that P. aeruginosa exotoxins S and T mediate in vitro the increase in protein permeability across lung endothelial cell monolayers via a RhoA-dependent mechanism. However, whether inhibition of RhoA would significantly attenuate P. aeruginosa-mediated lung injury in mice is unknown. METHODS: P. aeruginosa-induced paracellular protein permeability was measured across bovine lung endothelial and rat alveolar epithelial type II cell monolayers with I-albumin. Some cell monolayers were pretreated with RhoA inhibitor CGX0287 1 h before P. aeruginosa exposure. At 4 h after exposure, lung endothelial and epithelial permeability, bacterial counts, bronchoalveolar lavage fluid levels of keratinocyte-derived chemokine, myeloperoxidase activity, and alveolar fluid clearance were measured. Some mice were treated intraperitoneally with CGX0287 1 h before or after airspace instillation of P. aeruginosa. RESULTS: RhoA inhibition attenuated in vitro P. aeruginosa-mediated increase in lung endothelial and epithelial permeability to protein and in vivo the development of pulmonary edema and inhibition of alveolar fluid clearance associated with P. aeruginosa pneumonia. Furthermore, RhoA inhibition decreased the systemic dissemination of P. aeruginosa and neutrophil activity in the lung tissue observed after airspace instillation of these bacteria. CONCLUSIONS: The small GTPase RhoA plays a critical role in mediating lung injury associated with P. aeruginosa pneumonia in mice. Thus, transient blockade of RhoA could attenuate lung damage caused by P. aeruginosa in critically ill patients.

Acquired deficit of antithrombin and role of supplementation in septic patients during continuous veno-venous hemofiltration.

Continuous renal replacement therapy (CRRT) is widely used in the management of septic patients with acute renal failure (ARF). Short filter lifespan (<24 hours) is a major concern and may result of a procoagulating state. The aim of this study was to investigate the relationship between antithrombin (AT) deficit and early filter clotting, and whether supplementation of AT could increase filter lifespan. Two different methods for supplementation, bolus and continuous infusion were also compared. We conducted a two-center prospective study from March 2003 to May 2004. Twenty-seven patients with septic shock and ARF were included and treated by CRRT. Unfractionated heparin (UHF) was used for anticoagulation. The initial level of AT was low with a median level at 45.4% (16%-69%). Low AT activity was associated with shorter filter lifespan. Supplementation led to a longer filter lifespan (15.2-33.2 hours) (p < 0.05). Continuous infusion provided better results: 48.5 vs. 27.8 hours for bolus method. This study suggests that AT measurement should be considered in continuous veno-venous hemofiltration with clotting problems as supplementation could increase filter lifespan by more than 100%. Continuous infusion is preferable. Cost effectiveness should be evaluated shortly.