Key role of group v secreted phospholipase A2 in Th2 cytokine and dendritic cell-driven airway hyperresponsiveness and remodeling.

BACKGROUND: Previous work has shown that disruption of the gene for group X secreted phospholipase A2 (sPLA2-X) markedly diminishes airway hyperresponsiveness and remodeling in a mouse asthma model. With the large number of additional sPLA2s in the mammalian genome, the involvement of other sPLA2s in the asthma model is possible - in particular, the group V sPLA2 (sPLA2-V) that like sPLA2-X is highly active at hydrolyzing membranes of mammalian cells. METHODOLOGY AND PRINCIPAL FINDINGS: The allergen-driven asthma phenotype was significantly reduced in sPLA2-V-deficient mice but to a lesser extent than observed previously in sPLA2-X-deficient mice. The most striking difference observed between the sPLA2-V and sPLA2-X knockouts was the significant impairment of the primary immune response to the allergen ovalbumin (OVA) in the sPLA2-V(-/-) mice. The impairment in eicosanoid generation and dendritic cell activation in sPLA2-V(-/-) mice diminishes Th2 cytokine responses in the airways. CONCLUSIONS: This paper illustrates the diverse roles of sPLA2s in the immunopathogenesis of the asthma phenotype and directs attention to developing specific inhibitors of sPLA2-V as a potential new therapy to treat asthma and other allergic disorders.

Blockade of human group X secreted phospholipase A2 (GX-sPLA2)-induced airway inflammation and hyperresponsiveness in a mouse asthma model by a selective GX-sPLA2 inhibitor.

Group X (GX) phospholipase A(2), a member of a large group of secreted phospholipases A(2) (sPLA(2)s), has recently been demonstrated to play an important in vivo role in the release of arachidonic acid and subsequent formation of eicosanoids. In a Th2 cytokine-driven mouse asthma model, deficiency of mouse GX (mGX)-sPLA(2) significantly impairs development of the asthma phenotype. In this study, we generated mGX-sPLA(2)(-/-) mice with knock-in of human GX (hGX)-sPLA(2) (i.e. hGX-sPLA(2)(+/+) knock-in mice) to understand more fully the role of GX-sPLA(2) in these allergic pulmonary responses and to assess the effect of pharmacological blockade of the GX-sPLA(2)-mediated responses. Knock-in of hGX-sPLA(2) in mGX-sPLA(2)(-/-) mice restored the allergen-induced airway infiltration by inflammatory cells, including eosinophils, goblet cell metaplasia, and hyperresponsiveness to methacholine in the mGX-sPLA(2)-deficient mice. This knock-in mouse model enabled the use of a highly potent indole-based inhibitor of hGX-sPLA(2), RO061606 (which is ineffective against mGX-sPLA(2)), to assess the potential utility of GX-sPLA(2) blockade as a therapeutic intervention in asthma. Delivery of RO061606 via mini-osmotic pumps enabled the maintenance in vivo in the mouse asthma model of plasma inhibitor concentrations near 10 µm, markedly higher than the IC(50) for inhibition of hGX-sPLA(2) in vitro. RO061606 significantly decreased allergen-induced airway inflammation, mucus hypersecretion, and hyperresponsiveness in the hGX-sPLA(2)(+/+) knock-in mouse. Thus, development of specific hGX-sPLA(2) inhibitors may provide a new pharmacological opportunity for the treatment of patients with asthma.

Genetic ablation of phagocytic NADPH oxidase in mice limits TNFα-induced inflammation in the lungs but not other tissues.

In vitro and limited in vivo evidence suggests that reactive oxygen species derived from NADPH oxidases (NOX-ROS) play an important role in inflammatory responses by enhancing the activity of redox-sensitive cell signaling pathways and transcription factors. Here, we investigated the role of NOX-ROS in TNFα-induced acute inflammatory responses in vivo, using mice deficient in the gp91(phox) (NOX2) or p47(phox) subunits of NADPH oxidase. Age- and body weight-matched C57BL/6J wild-type (WT) and gp91(phox) or p47(phox) knockout mice were injected intraperitoneally with 50 µg TNFα/kg bw or saline vehicle control and sacrificed at various time points up to 24 h. Compared to WT mice, gp91(phox -/-) mice exhibited significantly diminished (P<0.05) TNFα-induced acute inflammatory responses in the lungs but not other tissues, including heart, liver, and kidney, as evidenced by decreased activation of the redox-sensitive transcription factor NF-κB, and decreased gene expression of interleukin (IL)-1ß, IL-6, TNFα, E-selectin, and other cellular adhesion molecules. Similar results were observed in p47(phox -/-) mice. Interestingly, decreased lung inflammation in knockout mice was accompanied by increased leukocyte infiltration into the lungs compared to other tissues. Our data suggest that phagocytic NOX-ROS signaling plays a critical role in promoting TNFα-induced, NF-κB-dependent acute inflammatory responses and tissue injury specifically in the lungs, which is effected by preferential leukocyte infiltration.

Inhibition of Wnt/beta-catenin/CREB binding protein (CBP) signaling reverses pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF)/usual interstitial pneumonia is a ravaging condition of progressive lung scarring and destruction. Anti-inflammatory therapies including corticosteroids have limited efficacy in this ultimately fatal disorder. An important unmet need is to identify new agents that interact with key molecular pathways involved in the pathogenesis of pulmonary fibrosis to prevent progression or reverse fibrosis in these patients. Because aberrant activation of the Wnt/beta-catenin signaling cascade occurs in lungs of patients with IPF, we have targeted this pathway for intervention in pulmonary fibrosis using ICG-001, a small molecule that specifically inhibits T-cell factor/beta-catenin transcription in a cyclic AMP response-element binding protein binding protein (CBP)-dependent fashion. ICG-001 selectively blocks the beta-catenin/CBP interaction without interfering with the beta-catenin/p300 interaction. We report here that ICG-001 (5 mg/kg per day) significantly inhibits beta-catenin signaling and attenuates bleomycin-induced lung fibrosis in mice, while concurrently preserving the epithelium. Administration of ICG-001 concurrent with bleomycin prevents fibrosis, and late administration is able to reverse established fibrosis and significantly improve survival. Because no effective treatment for IPF exists, selective inhibition of Wnt/beta-catenin-dependent transcription suggests a potential unique therapeutic approach for pulmonary fibrosis.

Moderate hypothermia (30 degrees C) maintains myocardial integrity and modifies response of cell survival proteins after reperfusion.

Hypothermia preserves myocardial function, promotes signaling for cell survival, and inhibits apoptotic pathways during 45-min reperfusion. We tested the hypothesis that signaling at the transcriptional level is followed by corresponding proteomic response and maintenance of structural integrity after 3-h reperfusion. Isolated hearts were Langendorff perfused and exposed to mild (I group; n = 6, 34 degrees C) or moderate (H group; n = 6, 30 degrees C) hypothermia during 120-min total ischemia with cardioplegic arrest and 180-min 37 degrees C reperfusion. Moderate hypothermia suppressed anaerobic metabolism during ischemia and significantly diminished left ventricular end-diastolic pressure at the end of ischemia from 52.7 +/- 3.3 (I group) to 1.8 +/- 0.9 (H group) mmHg. Unlike the I group, which showed poor cardiac function and high left ventricular pressure, the H group showed preservation of myocardial function, coronary flow, and oxygen consumption. Compared with normal control hearts without ischemia (n = 5), histological staining in the I group showed marked disarray and fragmentation of collagen network (score 4-5), while the H group showed preserved collagen integrity (score 0-1). The apoptosis-linked tumor suppressor protein p53 was expressed throughout the I group only (score 4-5). The H group produced elevated expression for hypoxia-inducible factor 1alpha and heme oxygenase 1, but minimally affected vascular endothelial growth factor expression. The H group also elevated expression for survival proteins peroxisomal proliferator-activated receptor-beta and Akt-1. These results show in a constant left ventricular volume model that moderate hypothermia (30 degrees C) decreases myocardial energy utilization during ischemia and subsequently promotes expression of proteins involved in cell survival, while inhibiting induction of p53 protein. These data also show that 34 degrees C proffers less protection and loss of myocardial integrity.

Importance of group X-secreted phospholipase A2 in allergen-induced airway inflammation and remodeling in a mouse asthma model.

Arachidonic acid metabolites, the eicosanoids, are key mediators of allergen-induced airway inflammation and remodeling in asthma. The availability of free arachidonate in cells for subsequent eicosanoid biosynthesis is controlled by phospholipase A(2)s (PLA(2)s), most notably cytosolic PLA(2)-alpha. 10 secreted PLA(2)s (sPLA(2)s) have also been identified, but their function in eicosanoid generation is poorly understood. We investigated the role of group X sPLA(2) (sPLA(2)-X), the sPLA(2) with the highest in vitro cellular phospholipolysis activity, in acute and chronic mouse asthma models in vivo. The lungs of sPLA(2)-X(-/-) mice, compared with those of sPLA(2)-X(+/+) littermates, had significant reduction in ovalbumin-induced infiltration by CD4(+) and CD8(+) T cells and eosinophils, goblet cell metaplasia, smooth muscle cell layer thickening, subepithelial fibrosis, and levels of T helper type 2 cell cytokines and eicosanoids. These data direct attention to sPLA(2)-X as a novel therapeutic target for asthma.

Reversal of allergen-induced airway remodeling by CysLT1 receptor blockade.

RATIONALE: Airway inflammation in asthma is accompanied by structural changes, including goblet cell metaplasia, smooth muscle cell layer thickening, and subepithelial fibrosis. This allergen-induced airway remodeling can be replicated in a mouse asthma model. OBJECTIVES: The study goal was to determine whether established airway remodeling in a mouse asthma model is reversible by administration of the cysteinyl leukotriene (CysLT)1 receptor antagonist montelukast, the corticosteroid dexamethasone, or the combination montelukast + dexamethasone. METHODS: BALB/c mice, sensitized by intraperitoneal ovalbumin (OVA) as allergen, received intranasal OVA periodically Days 14-73 and montelukast or dexamethasone or placebo from Days 73-163. MEASUREMENTS AND MAIN RESULTS: Allergen-induced trafficking of eosinophils into the bronchoalveolar lavage fluid and lung interstitium and airway goblet cell metaplasia, smooth muscle cell layer thickening, and subepithelial fibrosis present on Day 73 persisted at Day 163, 3 mo after the last allergen challenge. Airway hyperreactivity to methacholine observed on Day 73 in OVA-treated mice was absent on Day 163. In OVA-treated mice, airway eosinophil infiltration and goblet cell metaplasia were reduced by either montelukast or dexamethasone alone. Montelukast, but not dexamethasone, reversed the established increase in airway smooth muscle mass and subepithelial collagen deposition. By immunocytochemistry, CysLT1 receptor expression was significantly increased in airway smooth muscle cells in allergen-treated mice compared with saline-treated controls and was reduced by montelukast, but not dexamethasone, administration. CONCLUSIONS: These data indicate that established airway smooth muscle cell layer thickening and subepithelial fibrosis, key allergen-induced airway structural changes not modulated by corticosteroids, are reversible by CysLT1 receptor blockade therapy.