Alveolar CCN1 is associated with mechanical stretch and acute respiratory distress syndrome severity.

The cellular communication network factor 1 (CCN1) is a matricellular protein that can modulate multiple tissue responses, including inflammation and repair. We have previously shown that adenoviral overexpression of Ccn1 is sufficient to cause acute lung injury in mice. We hypothesized that CCN1 is present in the airspaces of lungs during the acute phase of lung injury, and higher concentrations are associated with acute respiratory distress syndrome (ARDS) severity. We tested this hypothesis by measuring 1) CCN1 in bronchoalveolar lavage fluid (BALF) and lung homogenates from mice subjected to ventilation-induced lung injury (VILI), 2) Ccn1 gene expression and protein levels in MLE-12 cells (alveolar epithelial cell line) subjected to mechanical stretch, and 3) CCN1 in BALF from mechanically ventilated humans with and without ARDS. BALF CCN1 concentrations and whole lung CCN1 protein levels were significantly increased in mice with VILI (n = 6) versus noninjured controls (n = 6). Ccn1 gene expression and CCN1 protein levels were increased in MLE-12 cells cultured under stretch conditions. Subjects with ARDS (n = 77) had higher BALF CCN1 levels compared with mechanically ventilated subjects without ARDS (n = 45) (P < 0.05). In subjects with ARDS, BALF CCN1 concentrations were associated with higher total protein, sRAGE, and worse [Formula: see text]/[Formula: see text] ratios (all P < 0.05). CCN1 is present in the lungs of mice and humans during the acute inflammatory phase of lung injury, and concentrations are higher in patients with increased markers of severity. Alveolar epithelial cells may be an important source of CCN1 under mechanical stretch conditions.

Fas activation alters tight junction proteins in acute lung injury.

Background:The acute respiratory distress syndrome (ARDS) is characterized by protein-rich oedema in the alveolar spaces, a feature in which Fas-mediated apoptosis of the alveolar epithelium has been involved. Objective:To determine whether Fas activation increases protein permeability by mechanisms involving disruption of the paracellular tight junction (TJ) proteins in the pulmonary alveoli. Methods: Protein permeability and the expression of TJ proteins were assessed in vivo in wild-type and Fas-deficient lpr mice 16 hours after the intratracheal instillation of recombinant human soluble Fas ligand (rh-sFasL), and at different time points in vitro in human pulmonary alveolar epithelial cells (HPAEpiC) exposed to rh-sFasL Results:Activation of the Fas pathway increased protein permeability in mouse lungs and altered the expression of the TJ proteins occludin and zonula occludens-1 in the alveolar-capillary membrane in vivo and in human alveolar epithelial cell monolayers in vitro. Blockade of caspase-3, but not inhibition of tyrosine kinase dependent pathways, prevented the alterations in TJ protein expression and permeability induced by the Fas/FasL system in human alveolar cell monolayers in vitro. We also observed that both the Fas-induced increase of protein permeability and disruption of TJ proteins occurred before cell death could be detected in the cell monolayers in vitro. Conclusion:Targeting caspase pathways could prevent the disruption of TJs and reduce the formation of lung oedema in the early stages of ARDS.

Endogenous secreted phospholipase A2 group X regulates cysteinyl leukotrienes synthesis by human eosinophils.

BACKGROUND: Phospholipase A2s mediate the rate-limiting step in the formation of eicosanoids such as cysteinyl leukotrienes (CysLTs). Group IVA cytosolic PLA2α (cPLA2α) is thought to be the dominant PLA2 in eosinophils; however, eosinophils also have secreted PLA2 (sPLA2) activity that has not been fully defined. OBJECTIVES: To examine the expression of sPLA2 group X (sPLA2-X) in eosinophils, the participation of sPLA2-X in the formation of CysLTs, and the mechanism by which sPLA2-X initiates the synthesis of CysLTs in eosinophils. METHODS: Peripheral blood eosinophils were obtained from volunteers with asthma and/or allergy. A rabbit polyclonal anti-sPLA2-X antibody identified sPLA2-X by Western blot. We used confocal microscopy to colocalize the sPLA2-X to intracellular structures. An inhibitor of sPLA2-X (ROC-0929) that does not inhibit other mammalian sPLA2s, as well as inhibitors of the mitogen-activated kinase cascade (MAPK) and cPLA2α, was used to examine the mechanism of N-formyl-methionyl-leucyl-phenylalanine (fMLP)-mediated formation of CysLT. RESULTS: Eosinophils express the mammalian sPLA2-X gene (PLA2G10). The sPLA2-X protein is located in the endoplasmic reticulum, golgi, and granules of eosinophils and moves to the granules and lipid bodies during fMLP-mediated activation. Selective sPLA2-X inhibition attenuated the fMLP-mediated release of arachidonic acid and CysLT formation by eosinophils. Inhibitors of p38, extracellular-signal-regulated kinases 1/2 (p44/42 MAPK), c-Jun N-terminal kinase, and cPLA2α also attenuated the fMLP-mediated formation of CysLT. The sPLA2-X inhibitor reduced the phosphorylation of p38 and extracellular-signal-regulated kinases 1/2 (p44/42 MAPK) as well as cPLA2α during cellular activation, indicating that sPLA2-X is involved in activating the MAPK cascade leading to the formation of CysLT via cPLA2α. We further demonstrate that sPLA2-X is activated before secretion from the cell during activation. Short-term priming with IL-13 and TNF/IL-1ß increased the expression of PLA2G10 by eosinophils. CONCLUSIONS: These results demonstrate that sPLA2-X plays a significant role in the formation of CysLTs by human eosinophils. The predominant role of the enzyme is the regulation of MAPK activation that leads to the phosphorylation of cPLA2α. The sPLA2-X protein is regulated by proteolytic cleavage, suggesting that an inflammatory environment may promote the formation of CysLTs through this mechanism. These results have important implications for the treatment of eosinophilic disorders such as asthma.

Symptom profiles and inflammatory markers in moderate to severe COPD.

BACKGROUND: Physical and psychological symptoms are the hallmark of patients' subjective perception of their illness. The purpose of this analysis was to determine if patients with COPD have distinctive symptom profiles and to examine the association of symptom profiles with systemic biomarkers of inflammation. METHODS: We conducted latent class analyses of three physical (dyspnea, fatigue, and pain) and two psychological symptoms (depression and anxiety) in 302 patients with moderate to severe COPD using baseline data from a longitudinal observational study of depression in COPD. Systemic inflammatory markers included IL1, IL8, IL10, IL12, IL13, INF, GM-CSF, TNF-α (levels >75thcentile was considered high); and CRP (levels >3 mg/L was considered high). Multinominal logistic regression models were used to examine the association between symptom classes and inflammation while adjusting for key socio-demographic and disease characteristics. RESULTS: We found that a 4-class model best fit the data: 1) low physical and psychological symptoms (26%, Low-Phys/Low-Psych), 2) low physical but moderate psychological symptoms (18%, Low-Phys/Mod Psych), 3) high physical but moderate psychological symptoms (25%, High-Phys/Mod Psych), and 4) high physical and psychological symptoms (30%, High-Phys/High Psych). Unadjusted analyses showed associations between symptom class with high levels of IL7, IL-8 (p ≤ .10) and CRP (p < .01). In the adjusted model, those with a high CRP level were less likely to be in the High-Phys/Mod-Psych class compared to the Low-Phys/Low-Psych (OR: 0.41, 95%CI 0.19, 0.90) and Low-Phys/Mod-Psych classes (OR: 0.35, 95%CI 0.16, 0.78); elevated CRP was associated with in increased odds of being in the High-Phys/High-Psych compared to the High-Phys/Mod-Psych class (OR: 2.22, 95%CI 1.08, 4.58). Younger age, having at least a college education, oxygen use and depression history were more prominent predictors of membership in the higher symptom classes. CONCLUSIONS: Patients with COPD can be classified into four distinct symptom classes based on five commonly co-occurring physical and psychological symptoms. Systemic biomarkers of inflammation were not associated with symptom class. Additional work to test the reliability of these symptom classes, their biological drivers and their validity for prognostication and tailoring therapy in larger and more diverse samples is needed. TRIAL REGISTRATION: Clinicaltrials.gov, NCT01074515 .

CYR61 (CCN1) overexpression induces lung injury in mice.

Cysteine-rich protein-61 (CYR61), also known as connective tissue growth factor, CYR61, and nephroblastoma overexpressed gene 1 (CCN1), is a heparin-binding protein member of the CCN family of matricellular proteins. Gene expression profiles showed that Cyr61 is upregulated in human acute lung injury (ALI), but its functional role is unclear. We hypothesized that CYR61 contributes to ALI in mice. First, we demonstrated that CYR61 expression increases after bleomycin-induced lung injury. We then used adenovirus-mediated gene transfer to determine whether CYR61 overexpression in the lungs was sufficient to cause ALI. Mice instilled with CYR61 adenovirus showed greater weight loss, increased bronchoalveolar lavage total neutrophil counts, increased protein concentrations, and increased mortality compared with mice instilled with empty-vector adenovirus. Immunohistochemical studies in lungs from humans with idiopathic pulmonary fibrosis revealed CYR61 expression on the luminal membrane of alveolar epithelial cells in areas of injury. We conclude that CYR61 is upregulated in ALI and that CYR61 overexpression exacerbates ALI in mice.

Airway epithelial regulation of pulmonary immune homeostasis and inflammation.

Recent genetic, structural and functional studies have identified the airway and lung epithelium as a key orchestrator of the immune response. Further, there is now strong evidence that epithelium dysfunction is involved in the development of inflammatory disorders of the lung. Here we review the characteristic immune responses that are orchestrated by the epithelium in response to diverse triggers such as pollutants, cigarette smoke, bacterial peptides, and viruses. We focus in part on the role of epithelium-derived interleukin (IL)-25, IL-33 and thymic stromal lymphopoietin (TSLP), as well as CC family chemokines as critical regulators of the immune response. We cite examples of the function of the epithelium in host defense and the role of epithelium dysfunction in the development of inflammatory diseases.

Effects of linezolid on suppressing in vivo production of staphylococcal toxins and improving survival outcomes in a rabbit model of methicillin-resistant Staphylococcus aureus necrotizing pneumonia.

BACKGROUND: Linezolid is recommended for treatment of pneumonia and other invasive infections caused by methicillin-resistant Staphylococcus aureus (MRSA). The premise underlying this recommendation is that linezolid inhibits in vivo production of potent staphylococcal exotoxins, including Panton-Valentine leukocidin (PVL) and α-hemolysin (Hla), although supporting evidence is lacking. METHODS: A rabbit model of necrotizing pneumonia using MRSA clone USA300 was used to compare therapeutic effects of linezolid (50 mg/kg 3 times/day) and vancomycin (30 mg/kg 2 times/day) administered 1.5, 4, and 9 hours after infection on host survival outcomes and in vivo bacterial toxin production. RESULTS: Mortality rates were 100% for untreated rabbits and 83%-100% for vancomycin-treated rabbits. In contrast, mortality rates were 25%, 50%, and 100% for rabbits treated with linezolid 1.5, 4, and 9 hours after infection, respectively. Compared with untreated and vancomycin-treated rabbits, improved survival of rabbits treated 1.5 hours after infection with linezolid was associated with a significant decrease in bacterial counts, suppressed bacterial production of PVL and Hla, and reduced production of the neutrophil-chemoattractant interleukin 8 in the lungs. CONCLUSIONS: Across the study interval, only early treatment with linezolid resulted in significant suppression of exotoxin synthesis and improved survival outcomes in a rabbit model of MRSA necrotizing pneumonia.

The Fas/FasL pathway impairs the alveolar fluid clearance in mouse lungs.

Alveolar epithelial damage is a critical event that leads to protein-rich edema in acute lung injury (ALI), but the mechanisms leading to epithelial damage are not completely understood. Cell death by necrosis and apoptosis occurs in alveolar epithelial cells in the lungs of patients with ALI. Fas activation induces apoptosis of alveolar epithelial cells, but its role in the formation of lung edema is unclear. The main goal of this study was to determine whether activation of the Fas/Fas ligand pathway in the lungs could alter the function of the lung epithelium, and the mechanisms involved. The results show that Fas activation alters the alveolar barrier integrity and impairs the ability of the lung alveolar epithelium to reabsorb fluid from the air spaces. This result was dependent on the presence of a normal Fas receptor and was not affected by inflammation induced by Fas activation. Alteration of the fluid transport properties of the alveolar epithelium was partially restored by ß-adrenergic stimulation. Fas activation also caused apoptosis of alveolar endothelial cells, but this effect was less pronounced than the effect on the alveolar epithelium. Thus, activation of the Fas pathway impairs alveolar epithelial function in mouse lungs by mechanisms involving caspase-dependent apoptosis, suggesting that targeting apoptotic pathways could reduce the formation of lung edema in ALI.

De novo design of highly selective miniprotein inhibitors of integrins αvß6 and αvß8.

The RGD (Arg-Gly-Asp)-binding integrins αvß6 and αvß8 are clinically validated cancer and fibrosis targets of considerable therapeutic importance. Compounds that can discriminate between the two closely related integrin proteins and other RGD integrins, stabilize specific conformational states, and have sufficient stability enabling tissue restricted administration could have considerable therapeutic utility. Existing small molecules and antibody inhibitors do not have all of these properties, and hence there is a need for new approaches. Here we describe a method for computationally designing hyperstable RGD-containing miniproteins that are highly selective for a single RGD integrin heterodimer and conformational state, and use this strategy to design inhibitors of αvß6 and αvß8 with high selectivity. The αvß6 and αvß8 inhibitors have picomolar affinities for their targets, and >1000-fold selectivity over other RGD integrins. CryoEM structures are within 0.6-0.7Å root-mean-square deviation (RMSD) to the computational design models; the designed αvß6 inhibitor and native ligand stabilize the open conformation in contrast to the therapeutic anti-αvß6 antibody BG00011 that stabilizes the bent-closed conformation and caused on-target toxicity in patients with lung fibrosis, and the αvß8 inhibitor maintains the constitutively fixed extended-closed αvß8 conformation. In a mouse model of bleomycin-induced lung fibrosis, the αvß6 inhibitor potently reduced fibrotic burden and improved overall lung mechanics when delivered via oropharyngeal administration mimicking inhalation, demonstrating the therapeutic potential of de novo designed integrin binding proteins with high selectivity.

De novo design of highly selective miniprotein inhibitors of integrins αvß6 and αvß8.

The RGD (Arg-Gly-Asp)-binding integrins αvß6 and αvß8 are clinically validated cancer and fibrosis targets of considerable therapeutic importance. Compounds that can discriminate between homologous αvß6 and αvß8 and other RGD integrins, stabilize specific conformational states, and have high thermal stability could have considerable therapeutic utility. Existing small molecule and antibody inhibitors do not have all these properties, and hence new approaches are needed. Here we describe a generalized method for computationally designing RGD-containing miniproteins selective for a single RGD integrin heterodimer and conformational state. We design hyperstable, selective αvß6 and αvß8 inhibitors that bind with picomolar affinity. CryoEM structures of the designed inhibitor-integrin complexes are very close to the computational design models, and show that the inhibitors stabilize specific conformational states of the αvß6 and the αvß8 integrins. In a lung fibrosis mouse model, the αvß6 inhibitor potently reduced fibrotic burden and improved overall lung mechanics, demonstrating the therapeutic potential of de novo designed integrin binding proteins with high selectivity.

Fas-deficient mice have impaired alveolar neutrophil recruitment and decreased expression of anti-KC autoantibody:KC complexes in a model of acute lung injury.

BACKGROUND: Exposure to mechanical ventilation enhances lung injury in response to various stimuli, such as bacterial endotoxin (LPS). The Fas/FasL system is a receptor ligand system that has dual pro-apoptotic and pro-inflammatory functions and has been implicated in the pathogenesis of lung injury. In this study we test the hypothesis that a functioning Fas/FasL system is required for the development of lung injury in mechanically ventilated mice. METHODS: C57BL/6 (B6) and Fas-deficient lpr mice were exposed to either intra-tracheal PBS followed by spontaneous breathing or intra-tracheal LPS followed by four hours mechanical ventilation with tidal volumes of 10 mL/kg, respiratory rate of 150 breaths per minute, inspired oxygen 0.21 and positive end expiratory pressure (PEEP) of 3 cm of water. RESULTS: Compared with the B6 mice, the lpr mice showed attenuation of the neutrophilic response as measured by decreased numbers of BAL neutrophils and lung myeloperoxidase activity. Interestingly, the B6 and lpr mice had similar concentrations of pro-inflammatory cytokines, including CXCL1 (KC), and similar measurements of permeability and apoptosis. However, the B6 mice showed greater deposition of anti-KC:KC immune complexes in the lungs, as compared with the lpr mice. CONCLUSIONS: We conclude that a functioning Fas/FasL system is required for full neutrophilic response to LPS in mechanically ventilated mice.

Febrile-range hyperthermia augments lipopolysaccharide-induced lung injury by a mechanism of enhanced alveolar epithelial apoptosis.

Fever is common in critically ill patients and is associated with worse clinical outcomes, including increased intensive care unit mortality. In animal models, febrile-range hyperthermia (FRH) worsens acute lung injury, but the mechanisms by which this occurs remain uncertain. We hypothesized that FRH augments the response of the alveolar epithelium to TNF-alpha receptor family signaling. We found that FRH augmented LPS-induced lung injury and increased LPS-induced mortality in mice. At 24 h, animals exposed to hyperthermia and LPS had significant increases in alveolar permeability without changes in inflammatory cells in bronchoalveolar lavage fluid or lung tissue as compared with animals exposed to LPS alone. The increase in alveolar permeability was associated with an increase in alveolar epithelial apoptosis and was attenuated by caspase inhibition with zVAD.fmk. At 48 h, the animals exposed to hyperthermia and LPS had an enhanced lung inflammatory response. In murine lung epithelial cell lines (MLE-15, LA-4) and in primary type II alveolar epithelial cells, FRH enhanced apoptosis in response to TNF-alpha but not Fas ligand. The increase in apoptosis was caspase-8 dependent and associated with suppression of NF-kappaB activity. The FRH-associated NF-kappaB suppression was not associated with persistence of IkappaB-alpha, suggesting that FRH-mediated suppression of NF-kappaB occurs by means other than alteration of IkappaB-alpha kinetics. These data show for the first time that FRH promotes lung injury in part by increasing lung epithelial apoptosis. The enhanced apoptotic response might relate to FRH-mediated suppression of NF-kappaB activity in the alveolar epithelium with a resultant increase in susceptibility to TNF-alpha-mediated cell death.

An official American Thoracic Society workshop report: features and measurements of experimental acute lung injury in animals.

Acute lung injury (ALI) is well defined in humans, but there is no agreement as to the main features of acute lung injury in animal models. A Committee was organized to determine the main features that characterize ALI in animal models and to identify the most relevant methods to assess these features. We used a Delphi approach in which a series of questionnaires were distributed to a panel of experts in experimental lung injury. The Committee concluded that the main features of experimental ALI include histological evidence of tissue injury, alteration of the alveolar capillary barrier, presence of an inflammatory response, and evidence of physiological dysfunction; they recommended that, to determine if ALI has occurred, at least three of these four main features of ALI should be present. The Committee also identified key "very relevant" and "somewhat relevant" measurements for each of the main features of ALI and recommended the use of least one "very relevant" measurement and preferably one or two additional separate measurements to determine if a main feature of ALI is present. Finally, the Committee emphasized that not all of the measurements listed can or should be performed in every study, and that measurements not included in the list are by no means "irrelevant." Our list of features and measurements of ALI is intended as a guide for investigators, and ultimately investigators should choose the particular measurements that best suit the experimental questions being addressed as well as take into consideration any unique aspects of the experimental design.

Fas activation in alveolar epithelial cells induces KC (CXCL1) release by a MyD88-dependent mechanism.

Activation of the Fas/Fas ligand (FasL) system is associated with activation of apoptotic and proinflammatory pathways that lead to the development of acute lung injury. Previous studies in chimeric mice and macrophage-depleted mice suggested that the main effector cell in Fas-mediated lung injury is not a myeloid cell, but likely an epithelial cell. The goal of this study was to determine whether epithelial cells release proinflammatory cytokines after Fas activation, and to identify the relevant pathways. Incubation of the murine alveolar epithelial cell line, MLE-12, with the Fas-activating monoclonal antibody, Jo2, resulted in release of the CXC chemokine, KC, in a dose-dependent manner. KC release was not prevented by the pan-caspase inhibitor, zVAD.fmk. Silencing of the adaptor protein, MyD88, with small interfering (si)RNA resulted in attenuation of KC release in response to Jo2. Fas activation resulted in phosphorylation of the mitogen-activated kinases extracellular signal-regulated kinase (ERK) and c-Jun-N-terminal kinase (JNK), and pharmacologic inhibition of ERK and JNK attenuated KC release in a dose-response manner. Similarly, primary human small airways epithelial cells released IL-8 in response to soluble FasL, and this was abrogated by inhibition of JNK and ERK. In vivo confirmatory studies showed that MyD88-null mice are protected from Fas-induced acute lung injury. In summary, we conclude that Fas induces KC release in MLE-12 cells by a mechanism requiring MyD88, mitogen-activated protein kinases, and likely activator protein-1.

Death receptors mediate the adverse effects of febrile-range hyperthermia on the outcome of lipopolysaccharide-induced lung injury.

We have shown that febrile-range hyperthermia enhances lung injury and mortality in mice exposed to inhaled LPS and is associated with increased TNF-α receptor activity, suppression of NF-κB activity in vitro, and increased apoptosis of alveolar epithelial cells in vivo. We hypothesized that hyperthermia enhances lung injury and mortality in vivo by a mechanism dependent on TNF receptor signaling. To test this, we exposed mice lacking the TNF-receptor family members TNFR1/R2 or Fas (TNFR1/R2(-/-) and lpr) to inhaled LPS with or without febrile-range hyperthermia. For comparison, we studied mice lacking IL-1 receptor activity (IL-1R(-/-)) to determine the role of inflammation on the effect of hyperthermia in vivo. TNFR1/R2(-/-) and lpr mice were protected from augmented alveolar permeability and mortality associated with hyperthermia, whereas IL-1R(-/-) mice were susceptible to augmented alveolar permeability but protected from mortality associated with hyperthermia. Hyperthermia decreased pulmonary concentrations of TNF-α and keratinocyte-derived chemokine after LPS in C57BL/6 mice and did not affect pulmonary inflammation but enhanced circulating markers of oxidative injury and nitric oxide metabolites. The data suggest that hyperthermia enhances lung injury by a mechanism that requires death receptor activity and is not directly associated with changes in inflammation mediated by hyperthermia. In addition, hyperthermia appears to enhance mortality by generating a systemic inflammatory response and not by a mechanism directly associated with respiratory failure. Finally, we observed that exposure to febrile-range hyperthermia converts a modest, survivable model of lung injury into a fatal syndrome associated with oxidative and nitrosative stress, similar to the systemic inflammatory response syndrome.