Jérémie Roux

Transforming Growth Factor-β1 Decreases Expression of the Epithelial Sodium Channel αENaC and Alveolar Epithelial Vectorial Sodium and Fluid Transport via an ERK1/2-dependent Mechanism

Acute lung injury (ALI) is characterized by the flooding of the alveolar airspaces with protein-rich edema fluid and diffuse alveolar damage. We have previously reported that transforming growth factor-β1 (TGF-β1) is a critical mediator of ALI after intratracheal administration of bleomycin or Escherichia coli endotoxin, at least in part due to effects on lung endothelial and alveolar epithelial permeability. In the present study, we hypothesized that TGF-β1 would also decrease vectorial ion and water transport across the distal lung epithelium. Therefore, we studied the effect of active TGF-β1 on 22Na+ uptake across monolayers of primary rat and human alveolar type II (ATII) cells. TGF-β1 significantly reduced the amiloride-sensitive fraction of 22Na+ uptake and fluid transport across monolayers of both rat and human ATII cells. TGF-β1 also significantly decreased αENaC mRNA and protein expression and inhibited expression of a luciferase reporter downstream of the αENaC promoter in lung epithelial cells. The inhibitory effect of TGF-β1 on sodium uptake and αENaC expression in ATII cells was mediated by activation of the MAPK, ERK1/2. Consistent with the in vitro results, TGF-β1 inhibited the amiloride-sensitive fraction of the distal airway epithelial fluid transport in an in vivo rat model at a dose that was not associated with any change in epithelial protein permeability. These data indicate that increased TGF-β1 activity in the distal airspaces during ALI promotes alveolar edema by reducing distal airway epithelial sodium and fluid clearance. This reduction in sodium and fluid transport is attributable in large part to a reduction in apical membrane αENaC expression mediated through an ERK1/2-dependent inhibition of the αENaC promoter activity.

Interleukin-1β Decreases Expression of the Epithelial Sodium Channel α-Subunit in Alveolar Epithelial Cells via a p38 MAPK-dependent Signaling Pathway

Acute lung injury (ALI) is a devastating syndrome characterized by diffuse alveolar damage, elevated airspace levels of pro-inflammatory cytokines, and flooding of the alveolar spaces with protein-rich edema fluid. Interleukin-1β (IL-1β) is one of the most biologically active cytokines in the distal airspaces of patients with ALI. IL-1β has been shown to increase lung epithelial and endothelial permeability. In this study, we hypothesized that IL-1β would decrease vectorial ion and water transport across the distal lung epithelium. Therefore, we measured the effects of IL-1β on transepithelial current, resistance, and sodium transport in primary cultures of alveolar epithelial type II (ATII) cells. IL-1β significantly reduced the amiloride-sensitive fraction of the transepithelial current and sodium transport across rat ATII cell monolayers. Moreover, IL-1β decreased basal and dexamethasone-induced epithelial sodium channel α-subunit (αENaC) mRNA levels and total and cell-surface protein expression. The inhibitory effect of IL-1β on αENaC expression was mediated by the activation of p38 MAPK in both rat and human ATII cells and was independent of the activation of αvβ6 integrin and transforming growth factor-β. These results indicate that IL-1β may contribute to alveolar edema in ALI by reducing distal lung epithelial sodium absorption. This reduction in ion and water transport across the lung epithelium is in large part due to a decrease in αENaC expression through p38 MAPK-dependent inhibition of αENaC promoter activity and to an alteration in ENaC trafficking to the apical membrane of ATII cells.

Interleukin-1β Causes Acute Lung Injury via αvβ5 and αvβ6 Integrin–Dependent Mechanisms

Interleukin (IL)-1&bgr; has previously been shown to be among the most biologically active cytokines in the lungs of patients with acute lung injury (ALI). Furthermore, there is experimental evidence that lung vascular permeability increases after short-term exposure to IL-1 protein, although the exact mechanism is unknown. Therefore, the objective of this study was to determine the mechanisms of IL-1&bgr;–mediated increase in lung vascular permeability and pulmonary edema following transient overexpression of this cytokine in the lungs by adenoviral gene transfer. Lung vascular permeability increased with intrapulmonary IL-1&bgr; production with a maximal effect 7 days after instillation of the adenovirus. Furthermore, inhibition of the &agr;v&bgr;6 integrin and/or transforming growth factor-&bgr; attenuated the IL-1&bgr;–induced ALI. The results of in vitro studies indicated that IL-1&bgr; caused the activation of transforming growth factor-&bgr; via RhoA/&agr;v&bgr;6 integrin–dependent mechanisms and the inhibition of the &agr;v&bgr;6 integrin and/or transforming growth factor-&bgr; signaling completely blocked the IL-1&bgr;–mediated protein permeability across alveolar epithelial cell monolayers. In addition, IL-1&bgr; increased protein permeability across lung endothelial cell monolayers via RhoA- and &agr;v&bgr;5 integrin–dependent mechanisms. The final series of in vivo experiments demonstrated that pretreatment with blocking antibodies to both the &agr;v&bgr;5 and &agr;v&bgr;6 integrins had an additive protective effect against IL-1&bgr;–induced ALI. In summary, these results demonstrate a critical role for the &agr;v&bgr;5/&bgr;6 integrins in mediating the IL-1&bgr;–induced ALI and indicate that these integrins could be a potentially attractive therapeutic target in ALI.

Reactive nitrogen species inhibit alveolar epithelial fluid transport after hemorrhagic shock in rats.

Our recent experimental work demonstrated that a neutrophil-dependent inflammatory response in the lung prevented the normal up-regulation of alveolar fluid clearance by catecholamines following hemorrhagic shock. In this study, we tested the hypothesis that the release of NO within the airspaces of the lung was responsible for the shock-mediated failure of the alveolar epithelium to respond to catecholamines in rats. Hemorrhagic shock was associated with an inducible NO synthase (iNOS)-dependent increase in the lung production of NO and a failure of the alveolar epithelium to up-regulate vectorial fluid transport in response to β-adrenergic agonists. Inhibition of iNOS restored the normal catecholamine-mediated up-regulation of alveolar liquid clearance. Airspace instillation of dibutyryl cAMP, a stable analog of cAMP, restored the normal fluid transport capacity of the alveolar epithelium after prolonged hemorrhagic shock, whereas direct stimulation of adenyl cyclase by forskolin had no effect. Pretreatment with pyrrolidine dithiocarbamate or sulfasalazine attenuated the iNOS-dependent production of NO in the lung and restored the normal up-regulation of alveolar fluid clearance by catecholamines after prolonged hemorrhagic shock. Based on in vitro studies with an alveolar epithelial cell line, A549 cells, the effect of sulfasalazine appeared to be mediated in part by inhibition of NF-κB activation, and the protective effect was mediated by the inhibition of IκBα protein degradation. In summary, these results provide the first in vivo evidence that NO, released within the airspaces of the lung probably secondary to the NF-κB-dependent activation of iNOS, is a major proximal inflammatory mediator that limits the rate of alveolar epithelial transport after prolonged hemorrhagic shock by directly impairing the function of membrane proteins involved in the β-adrenergic receptor-cAMP signaling pathway in alveolar epithelium.

Stress-Induced Inhibition of the NF-κB Signaling Pathway Results from the Insolubilization of the IκB Kinase Complex following Its Dissociation from Heat Shock Protein 90

Activation of the stress response attenuates proinflammatory responses by suppressing cytokine-stimulated activation of the NF-κB signaling pathway. In this study, we show that the activation of the cellular stress response, either by heat shock treatment or after exposure to sodium arsenite, leads to a transient inhibition of IκBα phosphorylation. Inhibition of IκBα phosphorylation after stress was associated with the detergent insolubilization of the upstream kinases, IκB kinase α (IKKα) and IκB kinase β, components involved in IκBα phosphorylation. Pretreatment of cells with glycerol, a chemical chaperone that reduces the extent of stress-induced protein denaturation, reduced the stress-dependent detergent insolubility of the IKK complex and restored the cytokine-stimulated phosphorylation of IκB. The stress-dependent insolubility of the IKK complex appeared reversible; as the cells recovered from the heat shock treatment, the IKK complex reappeared within the soluble fraction of cells and was again capable of mediating the phosphorylation of IκBα in response to added cytokines. Treatment of cells with geldanamycin, an inhibitor of heat shock protein 90 (Hsp90) function, also resulted in IKK detergent insolubility and proteasome-mediated degradation of the IKK complex. Furthermore, while IKKα coprecipitated with Hsp90 in control cells, coprecipitation of the two proteins was greatly reduced in those cells early after stress or following exposure to geldanamycin. Stress-induced transient insolubilization of the IκB kinase complex following its dissociation from Hsp90 represents a novel mechanism by which the activation of the stress response inhibits the NF-κB signaling pathway in response to proinflammatory stimuli.

The lectin-like domain of tumor necrosis factor improves lung function after rat lung transplantation—Potential role for a reduction in reactive oxygen species generation

Objective:To test the hypothesis that the lectin-like domain of tumor necrosis factor, mimicked by the TIP peptide, can improve lung function after unilateral orthotopic lung isotransplantation. Because of a lack of a specific treatment for ischemia reperfusion-mediated lung injury, accompanied by a disrupted barrier integrity and a dysfunctional alveolar liquid clearance, alternative therapies restoring these parameters after lung transplantation are required. Design:Prospective, randomized laboratory investigation. Setting:University-affiliated laboratory. Subjects:Adult female rats. Interventions:Tuberoinfundibular peptide, mimicking the lectin-like domain of tumor necrosis factor, mutant TIP peptide, N,N′-diacetylchitobiose/TIP peptide, and amiloride/TIP peptide were instilled intratracheally in the left lung immediately before the isotransplantation was performed. An additional group received an intravenous TIP peptide treatment, 1.5 mins before transplantation. Studies using isolated rat type II alveolar epithelial cell monolayers and ovine pulmonary endothelial cells were also performed. Measurements and Main Results:Intratracheal pretreatment of the transplantable left lung with the TIP peptide, but not with an inactive mutant TIP peptide, resulted in significantly improved oxygenation 24 hrs after transplantation. This treatment led to a significantly reduced neutrophil content in the lavage fluid. Both the effects on oxygenation and neutrophil infiltration were inhibited by the epithelial sodium channel blocker amiloride. The TIP peptide blunted reactive oxygen species production in pulmonary artery endothelial cells under hypoxia and reoxygenation and reduced reactive oxygen species content in the transplanted rat lungs in vivo. Ussing chamber experiments using monolayers of primary type II rat pneumocytes indicated that the primary site of action of the peptide was on the apical side of these cells. Conclusions:These data demonstrate that the TIP peptide significantly improves lung function after lung transplantation in the rat, in part, by reducing neutrophil content and reactive oxygen species generation. These studies suggest that the TIP peptide is a potential therapeutic agent against the ischemia reperfusion injury associated with lung transplantation.

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 aPCs 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 aPCs 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.

Transforming Growth Factor β1 Inhibits Cystic Fibrosis Transmembrane Conductance Regulator-dependent cAMP-stimulated Alveolar Epithelial Fluid Transport via a Phosphatidylinositol 3-Kinase-dependent Mechanism

Exogenous or endogenous β2-adrenergic receptor agonists enhance alveolar epithelial fluid transport via a cAMP-dependent mechanism that protects the lungs from alveolar flooding in acute lung injury. However, impaired alveolar fluid clearance is present in most of the patients with acute lung injury and is associated with increased mortality, although the mechanisms responsible for this inhibition of the alveolar epithelial fluid transport are not completely understood. Here, we found that transforming growth factor β1 (TGF-β1), a critical mediator of acute lung injury, inhibits β2-adrenergic receptor agonist-stimulated vectorial fluid and Cl− transport across primary rat and human alveolar epithelial type II cell monolayers. This inhibition is due to a reduction in the cystic fibrosis transmembrane conductance regulator activity and biosynthesis mediated by a phosphatidylinositol 3-kinase (PI3K)-dependent heterologous desensitization and down-regulation of the β2-adrenergic receptors. Consistent with these in vitro results, inhibition of the PI3K pathway or pretreatment with soluble chimeric TGF-β type II receptor restored β2-adrenergic receptor agonist-stimulated alveolar epithelial fluid transport in an in vivo model of acute lung injury induced by hemorrhagic shock in rats. The results demonstrate a novel role for TGF-β1 in impairing the β- adrenergic agonist-stimulated alveolar fluid clearance in acute lung injury, an effect that could be corrected by using PI3K inhibitors that are safe to use in humans.

Role of Small GTPases and αvβ5 Integrin in Pseudomonas aeruginosa–Induced Increase in Lung Endothelial Permeability

Pseudomonas aeruginosa is an opportunistic pathogen that can cause severe pneumonia associated with airspace flooding with protein-rich edema in critically ill patients. The type III secretion system is a major virulence factor and contributes to dissemination of P. aeruginosa. However, it is still unknown which particular bacterial toxin and which cellular pathways are responsible for the increase in lung endothelial permeability induced by P. aeruginosa. Thus, the first objective of this study was to determine the mechanisms by which this species causes an increase in lung endothelial permeability. The results showed that ExoS and ExoT, two of the four known P. aeruginosa type III cytotoxins, were primarily responsible for bacterium-induced increases in protein permeability across the lung endothelium via an inhibition of Rac1 and an activation of the RhoA signaling pathway. In addition, inhibition of the alphavbeta5 integrin, a central regulator of lung vascular permeability, prevented these P. aeruginosa-mediated increases in albumin flux due to endothelial permeability. Finally, prior activation of the stress protein response or adenoviral gene transfer of the inducible heat shock protein Hsp72 also inhibited the damaging effects of P. aeruginosa on the barrier function of lung endothelium. Taken together, these results demonstrate the critical role of the RhoA/alphavbeta5 integrin pathway in mediating P. aeruginosa-induced lung vascular permeability. In addition, activation of the stress protein response with pharmacologic inhibitors of Hsp90 may protect lungs against P. aeruginosa-induced permeability changes.

IL-8 inhibits cAMP-stimulated alveolar epithelial fluid transport via a GRK2/PI3K-dependent mechanism

Patients with acute lung injury (ALI) who retain maximal alveolar fluid clearance (AFC) have better clinical outcomes. Experimental and small clinical studies have shown that β2‐adrenergic receptor (β2AR) agonists enhance AFC via a cAMP‐dependent mechanism. However, two multicenter phase 3 clinical trials failed to show that β2AR agonists provide a survival advantage in patients with ALI. We hypothesized that IL‐8, an important mediator of ALI, directly antagonizes the alveolar epithelial response to β2AR agonists. Short‐circuit current and whole‐cell patch‐clamping experiments revealed that IL‐8 or its rat analog CINC‐1 decreases by 50% β2AR agonist‐stimulated vectorial Cl– and net fluid transport across rat and human alveolar epithelial type II cells via a reduction in the cystic fibrosis transmembrane conductance regulator activity and biosynthesis. This reduction was mediated by heterologous β2AR desensitization and down‐regulation (50%) via the G‐protein‐coupled receptor kinase 2 (GRK2)/PI3K signaling pathway. Inhibition of CINC‐1 restored β2AR agonist‐stimulated AFC in an experimental model of ALI in rats. Finally, consistent with the experimental results, high pulmonary edema fluid levels of IL‐8 (>4000 pg/ml) were associated with impaired AFC in patients with ALI. These results demonstrate a novel role for IL‐8 in inhibiting β2AR agonist‐stimulated alveolar epithelial fluid transport via GRK2/PI3K‐dependent mechanisms.—Roux, J., McNicholas, C. M., Carles, M., Goolaerts, A., Houseman, B. T., Dickinson, D. A., Iles, K. E., Ware, L. B., Matthay, M. A., Pittet, J.‐F. IL‐8 inhibits cAMP‐stimulated alveolar epithelial fluid transport via a GRK2/PI3K‐dependent mechanism. FASEB J. 27, 1095–1106 (2013). www.fasebj.org