Sphingosine kinase 1-specific inhibitor PF543 reduces goblet cell metaplasia of bronchial epithelium in an acute asthma model.

Sphingosine kinase 1 (SPHK1) has been shown to play a key role in the pathogenesis of asthma where SPHK1-generated sphingosine-1-phosphate (S1P) is known to mediate innate and adaptive immunity while promoting mast cell degranulation. Goblet cell metaplasia (GCM) contributes to airway obstruction in asthma and has been demonstrated in animal models. We investigated the role of PF543, a SPHK1-specific inhibitor, in preventing the pathogenesis of GCM using a murine (C57BL/6) model of allergen-induced acute asthma. Treatment with PF543 before triple allergen exposure (DRA: House dust mite, Ragweed pollen, and Aspergillus) reduced inflammation, eosinophilic response, and GCM followed by reduced airway hyperreactivity to intravenous methacholine. Furthermore, DRA exposure was associated with increased expression of SPHK1 in the airway epithelium which was reduced by PF543. DRA-induced reduction of acetylated α-tubulin in airway epithelium was associated with an increased expression of NOTCH2 and SPDEF which was prevented by PF543. In vitro studies using human primary airway epithelial cells showed that inhibition of SPHK1 using PF543 prevented an allergen-induced increase of both NOTCH2 and SPDEF. siRNA silencing of SPHK1 prevented the allergen-induced increase of both NOTCH2 and SPDEF. NOTCH2 silencing was associated with a reduction of SPDEF but not that of SPHK1 upon allergen exposure. Our studies demonstrate that inhibition of SPHK1 protected allergen-challenged airways by preventing GCM and airway hyperreactivity, associated with downregulation of the NOTCH2-SPDEF signaling pathway. This suggests a potential novel link between SPHK1, GCM, and airway remodeling in asthma.NEW & NOTEWORTHY The role of SPHK1-specific inhibitor, PF543, in preventing goblet cell metaplasia (GCM) and airway hyperreactivity (AHR) is established in an allergen-induced mouse model. This protection was associated with the downregulation of NOTCH2-SPDEF signaling pathway, suggesting a novel link between SPHK1, GCM, and AHR.

NOX4 Mediates Epithelial Cell Death in Hyperoxic Acute Lung Injury Through Mitochondrial Reactive Oxygen Species.

Management of acute respiratory distress involves O2 supplementation, which is lifesaving, but causes severe hyperoxic acute lung injury (HALI). NADPH oxidase (NOX) could be a major source of reactive oxygen species (ROS) in hyperoxia (HO). Epithelial cell death is a crucial step in the development of many lung diseases. Alveolar type II (AT2) cells are the metabolically active epithelial cells of alveoli that serve as a source of AT1 cells following lung injury. The aim of this study was to determine the possible role of AT2 epithelial cell NOX4 in epithelial cell death from HALI. Wild type (WT), Nox4 fl/fl (control), and Nox4 -/- Spc-Cre mice were exposed to room air (NO) or 95% O2 (HO) to investigate the structural and functional changes in the lung. C57BL/6J WT animals subjected to HO showed increased expression of lung NOX4 compared to NO. Significant HALI, increased bronchoalveolar lavage cell counts, increased protein levels, elevated proinflammatory cytokines and increased AT2 cell death seen in hyperoxic Nox4 fl/fl control mice were attenuated in HO-exposed Nox4 -/- Spc-Cre mice. HO-induced expression of NOX4 in MLE cells resulted in increased mitochondrial (mt) superoxide production and cell apoptosis, which was reduced in NOX4 siRNA silenced cells. This study demonstrates a novel role for epithelial cell NOX4 in accelerating lung epithelial cell apoptosis from HALI. Deletion of the Nox4 gene in AT2 cells or silencing NOX4 in lung epithelial cells protected the lungs from severe HALI with reduced apoptosis and decreased mt ROS production in HO. These results suggest NOX4 as a potential target for the treatment of HALI.

The Role of Sphingolipid Signaling in Oxidative Lung Injury and Pathogenesis of Bronchopulmonary Dysplasia.

Premature infants are born with developing lungs burdened by surfactant deficiency and a dearth of antioxidant defense systems. Survival rate of such infants has significantly improved due to advances in care involving mechanical ventilation and oxygen supplementation. However, a significant subset of such survivors develops the chronic lung disease, Bronchopulmonary dysplasia (BPD), characterized by enlarged, simplified alveoli and deformed airways. Among a host of factors contributing to the pathogenesis is oxidative damage induced by exposure of the developing lungs to hyperoxia. Recent data indicate that hyperoxia induces aberrant sphingolipid signaling, leading to mitochondrial dysfunction and abnormal reactive oxygen species (ROS) formation (ROS). The role of sphingolipids such as ceramides and sphingosine 1-phosphate (S1P), in the development of BPD emerged in the last decade. Both ceramide and S1P are elevated in tracheal aspirates of premature infants of <32 weeks gestational age developing BPD. This was faithfully reflected in the murine models of hyperoxia and BPD, where there is an increased expression of sphingolipid metabolites both in lung tissue and bronchoalveolar lavage. Treatment of neonatal pups with a sphingosine kinase1 specific inhibitor, PF543, resulted in protection against BPD as neonates, accompanied by improved lung function and reduced airway remodeling as adults. This was accompanied by reduced mitochondrial ROS formation. S1P receptor1 induced by hyperoxia also aggravates BPD, revealing another potential druggable target in this pathway for BPD. In this review we aim to provide a detailed description on the role played by sphingolipid signaling in hyperoxia induced lung injury and BPD.

Sphingosine kinase 1 regulates lysyl oxidase through STAT3 in hyperoxia-mediated neonatal lung injury.

INTRODUCTION: Neonatal lung injury as a consequence of hyperoxia (HO) therapy and ventilator care contribute to the development of bronchopulmonary dysplasia (BPD). Increased expression and activity of lysyl oxidase (LOX), a key enzyme that cross-links collagen, was associated with increased sphingosine kinase 1 (SPHK1) in human BPD. We, therefore, examined closely the link between LOX and SPHK1 in BPD. METHOD: The enzyme expression of SPHK1 and LOX were assessed in lung tissues of human BPD using immunohistochemistry and quantified (Halo). In vivo studies were based on Sphk1-/- and matched wild type (WT) neonatal mice exposed to HO while treated with PF543, an inhibitor of SPHK1. In vitro mechanistic studies used human lung microvascular endothelial cells (HLMVECs). RESULTS: Both SPHK1 and LOX expressions were increased in lungs of patients with BPD. Tracheal aspirates from patients with BPD had increased LOX, correlating with sphingosine-1-phosphate (S1P) levels. HO-induced increase of LOX in lungs were attenuated in both Sphk1-/- and PF543-treated WT mice, accompanied by reduced collagen staining (sirius red). PF543 reduced LOX activity in both bronchoalveolar lavage fluid and supernatant of HLMVECs following HO. In silico analysis revealed STAT3 as a potential transcriptional regulator of LOX. In HLMVECs, following HO, ChIP assay confirmed increased STAT3 binding to LOX promoter. SPHK1 inhibition reduced phosphorylation of STAT3. Antibody to S1P and siRNA against SPNS2, S1P receptor 1 (S1P1) and STAT3 reduced LOX expression. CONCLUSION: HO-induced SPHK1/S1P signalling axis plays a critical role in transcriptional regulation of LOX expression via SPNS2, S1P1 and STAT3 in lung endothelium.

A Minimally Invasive Method for Intratracheal Instillation of Drugs in Neonatal Rodents to Treat Lung Disease.

Treatment of neonatal rodent with drugs instilled directly into the trachea could serve as a valuable tool to study the impact of a locally administered drug. This has direct translational impact because surfactant and drugs are administered locally into the lungs. Though the literature has many publications describing minimally invasive transoral intubation of adult mice and rats in therapeutic experiments, this approach in neonatal rat pups is lacking. The small size of orotracheal region/pharynx in the pups makes visualization of laryngeal lumen (vocal cords) difficult, contributing to the variable success rate of intratracheal drug delivery. We hereby demonstrate effective oral intubation of neonatal rat pup - a technique that is non-traumatic and minimally-invasive, so that it can be used for serial administration of drugs. We used an operating otoscope with an illumination system and a magnifying lens to visualize the tracheal opening of the rat neonates. The drug is then instilled using a 1 mL syringe connected to a pipette tip. The accuracy of the delivery method was demonstrated using Evans blue dye administration. This method is easy to get trained in and could serve as an effective way to instill drugs into trachea. This method could also be used for administration of inoculum or agents to simulate disease conditions in animals and, also, for cell-based treatment strategies for various lung diseases.

Hyperoxia-induced S1P1 signaling reduced angiogenesis by suppression of TIE-2 leading to experimental bronchopulmonary dysplasia.

INTRODUCTION: We have earlier shown that hyperoxia (HO)-induced sphingosine kinase 1 (SPHK1)/sphingosine-1-phosphate (S1P) signaling contribute to bronchopulmonary dysplasia (BPD). S1P acts through G protein-coupled receptors, S1P1 through S1P5. Further, we noted that heterozygous deletion of S1pr1 ameliorated the HO-induced BPD in the murine model. The mechanism by which S1P1 signaling contributes to HO-induced BPD was explored. METHODS: S1pr1+/+ and S1pr1+/- mice pups were exposed to either room air (RA) or HO (75% oxygen) for 7 days from PN 1-7. Lung injury and alveolar simplification was evaluated. Lung protein expression was determined by Western blotting and immunohistochemistry (IHC). In vitro experiments were performed using human lung microvascular endothelial cells (HLMVECs) with S1P1 inhibitor, NIBR0213 to interrogate the S1P1 signaling pathway. RESULTS: HO increased the expression of S1pr1 gene as well as S1P1 protein in both neonatal lungs and HLMVECs. The S1pr1+/- neonatal mice showed significant protection against HO-induced BPD which was accompanied by reduced inflammation markers in the bronchoalveolar lavage fluid. HO-induced reduction in ANG-1, TIE-2, and VEGF was rescued in S1pr1+/- mouse, accompanied by an improvement in the number of arterioles in the lung. HLMVECs exposed to HO increased the expression of KLF-2 accompanied by reduced expression of TIE-2, which was reversed with S1P1 inhibition. CONCLUSION: HO induces S1P1 followed by reduced expression of angiogenic factors. Reduction of S1P1 signaling restores ANG-1/ TIE-2 signaling leading to improved angiogenesis and alveolarization thus protecting against HO-induced neonatal lung injury.

NOX4 Mediates Pseudomonas aeruginosa-Induced Nuclear Reactive Oxygen Species Generation and Chromatin Remodeling in Lung Epithelium.

Pseudomonas aeruginosa (PA) infection increases reactive oxygen species (ROS), and earlier, we have shown a role for NADPH oxidase-derived ROS in PA-mediated lung inflammation and injury. Here, we show a role for the lung epithelial cell (LEpC) NOX4 in PA-mediated chromatin remodeling and lung inflammation. Intratracheal administration of PA to Nox4flox/flox mice for 24 h caused lung inflammatory injury; however, epithelial cell-deleted Nox4 mice exhibited reduced lung inflammatory injury, oxidative stress, secretion of pro-inflammatory cytokines, and decreased histone acetylation. In LEpCs, NOX4 was localized both in the cytoplasmic and nuclear fractions, and PA stimulation increased the nuclear NOX4 expression and ROS production. Downregulation or inhibition of NOX4 and PKC δ attenuated the PA-induced nuclear ROS. PA-induced histone acetylation was attenuated by Nox4-specific siRNA, unlike Nox2. PA stimulation increased HDAC1/2 oxidation and reduced HDAC1/2 activity. The PA-induced oxidation of HDAC2 was attenuated by N-acetyl-L-cysteine and siRNA specific for Pkc δ, Sphk2, and Nox4. PA stimulated RAC1 activation in the nucleus and enhanced the association between HDAC2 and RAC1, p-PKC δ, and NOX4 in LEpCs. Our results revealed a critical role for the alveolar epithelial NOX4 in mediating PA-induced lung inflammatory injury via nuclear ROS generation, HDAC1/2 oxidation, and chromatin remodeling.

The Sphingosine Kinase 1 Inhibitor, PF543, Mitigates Pulmonary Fibrosis by Reducing Lung Epithelial Cell mtDNA Damage and Recruitment of Fibrogenic Monocytes.

Idiopathic pulmonary fibrosis (IPF) is a chronic disease for which novel approaches are urgently required. We reported increased sphingosine kinase 1 (SPHK1) in IPF lungs and that SPHK1 inhibition using genetic and pharmacologic approaches reduces murine bleomycin-induced pulmonary fibrosis. We determined whether PF543, a specific SPHK1 inhibitor post bleomycin or asbestos challenge mitigates lung fibrosis by reducing mitochondrial (mt) DNA damage and pro-fibrotic monocyte recruitment-both are implicated in the pathobiology of pulmonary fibrosis. Bleomycin (1.5 U/kg), crocidolite asbestos (100 µg/50 µL) or controls was intratracheally instilled in Wild-Type (C57Bl6) mice. PF543 (1 mg/kg) or vehicle was intraperitoneally injected once every two days from day 7-21 following bleomycin and day 14-21 or day 30-60 following asbestos. PF543 reduced bleomycin- and asbestos-induced pulmonary fibrosis at both time points as well as lung expression of profibrotic markers, lung mtDNA damage, and fibrogenic monocyte recruitment. In contrast to human lung fibroblasts, asbestos augmented lung epithelial cell (MLE) mtDNA damage and PF543 was protective. Post-exposure PF543 mitigates pulmonary fibrosis in part by reducing lung epithelial cell mtDNA damage and monocyte recruitment. We reason that SPHK1 signaling may be an innovative therapeutic target for managing patients with IPF and other forms of lung fibrosis.

Lysocardiolipin acyltransferase regulates NSCLC cell proliferation and migration by modulating mitochondrial dynamics.

Lysocardiolipin acyltransferase (LYCAT), a cardiolipin (CL)-remodeling enzyme, is crucial for maintaining normal mitochondrial function and vascular development. Despite the well-characterized role for LYCAT in the regulation of mitochondrial dynamics, its involvement in lung cancer, if any, remains incompletely understood. In this study, in silico analysis of TCGA lung cancer data sets revealed a significant increase in LYCAT expression, which was later corroborated in human lung cancer tissues and immortalized lung cancer cell lines via indirect immunofluorescence and immunoblotting, respectively. Stable knockdown of LYCAT in NSCLC cell lines not only reduced CL and increased monolyso-CL levels but also reduced in vivo tumor growth, as determined by xenograft studies in athymic nude mice. Furthermore, blocking LYCAT activity using a LYCAT mimetic peptide attenuated cell migration, suggesting a novel role for LYCAT activity in promoting NSCLC. Mechanistically, the pro-proliferative effects of LYCAT were mediated by an increase in mitochondrial fusion and a G1/S cell cycle transition, both of which are linked to increased cell proliferation. Taken together, these results demonstrate a novel role for LYCAT in promoting NSCLC and suggest that targeting LYCAT expression or activity in NSCLC may provide new avenues for the therapeutic treatment of lung cancer.

Neonatal therapy with PF543, a sphingosine kinase 1 inhibitor, ameliorates hyperoxia-induced airway remodeling in a murine model of bronchopulmonary dysplasia.

Hyperoxia (HO)-induced lung injury contributes to bronchopulmonary dysplasia (BPD) in preterm newborns. Intractable wheezing seen in BPD survivors is associated with airway remodeling (AWRM). Sphingosine kinase 1 (SPHK1)/sphingosine-1-phosphate (S1P) signaling promotes HO-mediated neonatal BPD; however, its role in the sequela of AWRM is not known. We noted an increased concentration of S1P in tracheal aspirates of neonatal infants with severe BPD, and earlier, demonstrated that Sphk1-/- mice showed protection against HO-induced BPD. The role of SPHK1/S1P in promoting AWRM following exposure of neonates to HO was investigated in a murine model. Therapy using PF543, the specific SPHK1 inhibitor, during neonatal HO reduced alveolar simplification followed by reduced AWRM in adult mice. This was associated with reduced airway hyperreactivity to intravenous methacholine. Neonatal HO exposure was associated with increased expression of SPHK1 in lung tissue of adult mice, which was reduced with PF543 therapy in the neonatal stage. This was accompanied by amelioration of HO-induced reduction of E-cadherin in airway epithelium. This may be suggestive of arrested partial epithelial mesenchymal transition (EMT) induced by HO. In vitro studies using human primary airway epithelial cells (HAEpCs) showed that SPHK1 inhibition or deletion restored HO-induced reduction in E-cadherin and reduced formation of mitochondrial reactive oxygen species (mtROS). Blocking mtROS with MitoTempo attenuated HO-induced partial EMT of HAEpCs. These results collectively support a therapeutic role for PF543 in preventing HO-induced BPD in neonates and the long-term sequela of AWRM, thus conferring a long-term protection resulting in improved lung development and function.

Sphingosine Kinase 1/S1P Signaling Contributes to Pulmonary Fibrosis by Activating Hippo/YAP Pathway and Mitochondrial Reactive Oxygen Species in Lung Fibroblasts.

The sphingosine kinase 1 (SPHK1)/sphingosine-1-phosphate (S1P) signaling axis is emerging as a key player in the development of idiopathic pulmonary fibrosis (IPF) and bleomycin (BLM)-induced lung fibrosis in mice. Recent evidence implicates the involvement of the Hippo/Yes-associated protein (YAP) 1 pathway in lung diseases, including IPF, but its plausible link to the SPHK1/S1P signaling pathway is unclear. Herein, we demonstrate the increased co-localization of YAP1 with the fibroblast marker FSP1 in the lung fibroblasts of BLM-challenged mice, and the genetic deletion of Sphk1 in mouse lung fibroblasts (MLFs) reduced YAP1 localization in fibrotic foci. The PF543 inhibition of SPHK1 activity in mice attenuated YAP1 co-localization with FSP1 in lung fibroblasts. In vitro, TGF-ß stimulated YAP1 translocation to the nucleus in primary MLFs, and the deletion of Sphk1 or inhibition with PF543 attenuated TGF-ß-mediated YAP1 nuclear localization. Moreover, the PF543 inhibition of SPHK1, or the verteporfin inhibition of YAP1, decreased the TGF-ß- or BLM-induced mitochondrial reactive oxygen species (mtROS) in human lung fibroblasts (HLFs) and the expression of fibronectin (FN) and alpha-smooth muscle actin (α-SMA). Furthermore, scavenging mtROS with MitoTEMPO attenuated the TGF-ß-induced expression of FN and α-SMA. The addition of the S1P antibody to HLFs reduced TGF-ß- or S1P-mediated YAP1 activation, mtROS, and the expression of FN and α-SMA. These results suggest a role for SPHK1/S1P signaling in TGF-ß-induced YAP1 activation and mtROS generation, resulting in fibroblast activation, a critical driver of pulmonary fibrosis.

Advancements in understanding the role of lysophospholipids and their receptors in lung disorders including bronchopulmonary dysplasia.

Bronchopulmonary dysplasia (BPD) is a devastating chronic neonatal lung disease leading to serious adverse consequences. Nearly 15 million babies are born preterm accounting for >1 in 10 births globally. The aetiology of BPD is multifactorial and the survivors suffer lifelong respiratory morbidity. Lysophospholipids (LPL), which include sphingosine-1-phosphate (S1P), and lysophosphatidic acid (LPA) are both naturally occurring bioactive lipids involved in a variety of physiological and pathological processes such as cell survival, death, proliferation, migration, immune responses and vascular development. Altered LPL levels have been observed in a number of lung diseases including BPD, which underscores the importance of these signalling lipids under normal and pathophysiological situations. Due to the paucity of information related to LPLs in BPD, most of the ideas related to BPD and LPL are speculative. This article is intended to promote discussion and generate hypotheses, in addition to the limited review of information related to BPD already established in the literature.

S1P and plasmalogen derived fatty aldehydes in cellular signaling and functions.

Long-chain fatty aldehydes are present in low concentrations in mammalian cells and serve as intermediates in the interconversion between fatty acids and fatty alcohols. The long-chain fatty aldehydes are generated by enzymatic hydrolysis of 1-alkyl-, and 1-alkenyl-glycerophospholipids by alkylglycerol monooxygenase, plasmalogenase or lysoplasmalogenase while hydrolysis of sphingosine-1-phosphate (S1P) by S1P lyase generates trans ∆2-hexadecenal (∆2-HDE). Additionally, 2-chloro-, and 2-bromo- fatty aldehydes are produced from plasmalogens or lysoplasmalogens by hypochlorous, and hypobromous acid generated by activated neutrophils and eosinophils, respectively while 2-iodofatty aldehydes are produced by excess iodine in thyroid glands. The 2-halofatty aldehydes and ∆2-HDE activated JNK signaling, BAX, cytoskeletal reorganization and apoptosis in mammalian cells. Further, 2-chloro- and 2-bromo-fatty aldehydes formed GSH and protein adducts while ∆2-HDE formed adducts with GSH, deoxyguanosine in DNA and proteins such as HDAC1 in vitro. ∆2-HDE also modulated HDAC activity and stimulated H3 and H4 histone acetylation in vitro with lung epithelial cell nuclear preparations. The α-halo fatty aldehydes elicited endothelial dysfunction, cellular toxicity and tissue damage. Taken together, these investigations suggest a new role for long-chain fatty aldehydes as signaling lipids, ability to form adducts with GSH, proteins such as HDACs and regulate cellular functions.

Colony-stimulating factor 1 and its receptor are new potential therapeutic targets for allergic asthma.

BACKGROUND: A new approach targeting aeroallergen sensing in the early events of mucosal immunity could have greater benefit. The CSF1-CSF1R pathway has a critical role in trafficking allergens to regional lymph nodes through activating dendritic cells. Intervention in this pathway could prevent allergen sensitization and subsequent Th2 allergic inflammation. OBJECTIVE: To examine the therapeutic effectiveness of CSF1 and CSF1R inhibition for blocking the dendritic cell function of sensing aeroallergens. METHODS: We adopted a model of chronic asthma induced by a panel of three naturally occurring allergens and novel delivery system of CSF1R inhibitor encapsulated nanoprobe. RESULTS: Selective depletion of CSF1 in airway epithelial cells abolished the production of allergen-reactive IgE, resulting in prevention of new asthma development as well as reversal of established allergic lung inflammation. CDPL-GW nanoprobe containing GW2580, a selective CSF1R inhibitor, showed favorable pharmacokinetics for inhalational treatment and intranasal insufflation delivery of CDPL-GW nanoprobe ameliorated asthma pathologies including allergen-specific serum IgE production, allergic lung and airway inflammation and airway hyper-responsiveness (AHR) with minimal pulmonary adverse reaction. CONCLUSION: The inhibition of the CSF1-CSF1R signaling pathway effectively suppresses sensitization to aeroallergens and consequent allergic lung inflammation in a murine model of chronic asthma. CSF1R inhibition is a promising new target for the treatment of allergic asthma.

Genetic deletion of Sphk2 confers protection against Pseudomonas aeruginosa mediated differential expression of genes related to virulent infection and inflammation in mouse lung.

BACKGROUND: Pseudomonas aeruginosa (PA) is an opportunistic Gram-negative bacterium that causes serious life threatening and nosocomial infections including pneumonia. PA has the ability to alter host genome to facilitate its invasion, thus increasing the virulence of the organism. Sphingosine-1- phosphate (S1P), a bioactive lipid, is known to play a key role in facilitating infection. Sphingosine kinases (SPHK) 1&2 phosphorylate sphingosine to generate S1P in mammalian cells. We reported earlier that Sphk2-/- mice offered significant protection against lung inflammation, compared to wild type (WT) animals. Therefore, we profiled the differential expression of genes between the protected group of Sphk2-/- and the wild type controls to better understand the underlying protective mechanisms related to the Sphk2 deletion in lung inflammatory injury. Whole transcriptome shotgun sequencing (RNA-Seq) was performed on mouse lung tissue using NextSeq 500 sequencing system. RESULTS: Two-way analysis of variance (ANOVA) analysis was performed and differentially expressed genes following PA infection were identified using whole transcriptome of Sphk2-/- mice and their WT counterparts. Pathway (PW) enrichment analyses of the RNA seq data identified several signaling pathways that are likely to play a crucial role in pneumonia caused by PA such as those involved in: 1. Immune response to PA infection and NF-κB signal transduction; 2. PKC signal transduction; 3. Impact on epigenetic regulation; 4. Epithelial sodium channel pathway; 5. Mucin expression; and 6. Bacterial infection related pathways. Our genomic data suggests a potential role for SPHK2 in PA-induced pneumonia through elevated expression of inflammatory genes in lung tissue. Further, validation by RT-PCR on 10 differentially expressed genes showed 100% concordance in terms of vectoral changes as well as significant fold change. CONCLUSION: Using Sphk2-/- mice and differential gene expression analysis, we have shown here that S1P/SPHK2 signaling could play a key role in promoting PA pneumonia. The identified genes promote inflammation and suppress others that naturally inhibit inflammation and host defense. Thus, targeting SPHK2/S1P signaling in PA-induced lung inflammation could serve as a potential therapy to combat PA-induced pneumonia.

Pseudomonas aeruginosa stimulates nuclear sphingosine-1-phosphate generation and epigenetic regulation of lung inflammatory injury.

INTRODUCTION: Dysregulated sphingolipid metabolism has been implicated in the pathogenesis of various pulmonary disorders. Nuclear sphingosine-1-phosphate (S1P) has been shown to regulate histone acetylation, and therefore could mediate pro-inflammatory genes expression. METHODS: Profile of sphingolipid species in bronchoalveolar lavage fluids and lung tissue of mice challenged with Pseudomonas aeruginosa (PA) was investigated. The role of nuclear sphingosine kinase (SPHK)2 and S1P in lung inflammatory injury by PA using genetically engineered mice was determined. RESULTS: Genetic deletion of Sphk2, but not Sphk1, in mice conferred protection from PA-mediated lung inflammation. PA infection stimulated phosphorylation of SPHK2 and its localisation in epithelial cell nucleus, which was mediated by protein kinase C (PKC) δ. Inhibition of PKC δ or SPHK2 activity reduced PA-mediated acetylation of histone H3 and H4, which was necessary for the secretion of pro-inflammatory cytokines, interleukin-6 and tumour necrosis factor-α. The clinical significance of the findings is supported by enhanced nuclear localisation of p-SPHK2 in the epithelium of lung specimens from patients with cystic fibrosis (CF). CONCLUSIONS: Our studies define a critical role for nuclear SPHK2/S1P signalling in epigenetic regulation of bacterial-mediated inflammatory lung injury. Targeting SPHK2 may represent a potential strategy to reduce lung inflammatory pulmonary disorders such as pneumonia and CF.

Novel peptide nanoparticle-biased antagonist of CCR3 blocks eosinophil recruitment and airway hyperresponsiveness.

BACKGROUND: Chemokine signaling through CCR3 is a key regulatory pathway for eosinophil recruitment into tissues associated with allergic inflammation and asthma. To date, none of the CCR3 antagonists have shown efficacy in clinical trials. One reason might be their unbiased mode of inhibition that prevents receptor internalization, leading to drug tolerance. OBJECTIVE: We sought to develop a novel peptide nanoparticle CCR3 inhibitor (R321) with a biased mode of inhibition that would block G protein signaling but enable or promote receptor internalization. METHODS: Self-assembly of R321 peptide into nanoparticles and peptide binding to CCR3 were analyzed by means of dynamic light scattering and nuclear magnetic resonance. Inhibitory activity on CCR3 signaling was assessed in vitro by using flow cytometry, confocal microscopy, and Western blot analysis in a CCR3+ eosinophil cell line and blood eosinophils. In vivo effects of R321 were assessed by using a triple-allergen mouse asthma model. RESULTS: R321 self-assembles into nanoparticles and binds directly to CCR3, altering receptor function. Half-maximal inhibitory concentration values for eotaxin-induced chemotaxis of blood eosinophils are in the low nanomolar range. R321 inhibits only the early phase of extracellular signal-regulated kinase 1/2 activation and not the late phase generally associated with ß-arrestin recruitment and receptor endocytosis, promoting CCR3 internalization and degradation. In vivo R321 effectively blocks eosinophil recruitment into the blood, lungs, and airways and prevents airway hyperresponsiveness in a mouse eosinophilic asthma model. CONCLUSIONS: R321 is a potent and selective antagonist of the CCR3 signaling cascade. Inhibition through a biased mode of antagonism might hold significant therapeutic promise by eluding the formation of drug tolerance.

Sphingolipids Signaling in Lamellipodia Formation and Enhancement of Endothelial Barrier Function.

Sphingolipids, first described in the brain in 1884, are important structural components of biological membranes of all eukaryotic cells. In recent years, several lines of evidence support the critical role of sphingolipids such as sphingosine, sphingosine-1-phosphate (S1P), and ceramide as anti- or pro-inflammatory bioactive lipid mediators in a variety of human pathologies including pulmonary and vascular disorders. Among the sphingolipids, S1P is a naturally occurring agonist that exhibits potent barrier enhancing property in the endothelium by signaling via G protein-coupled S1P1 receptor. S1P, S1P analogs, and other barrier enhancing agents such as HGF, oxidized phospholipids, and statins also utilize the S1P/S1P1 signaling pathway to generate membrane protrusions or lamellipodia, which have been implicated in resealing of endothelial gaps and maintenance of barrier integrity. A better understanding of sphingolipids mediated regulation of lamellipodia formation and barrier enhancement of the endothelium will be critical for the development of sphingolipid-based therapies to alleviate pulmonary disorders such as sepsis-, radiation-, and mechanical ventilation-induced acute lung injury.

Sphingolipids in Ventilator Induced Lung Injury: Role of Sphingosine-1-Phosphate Lyase.

Mechanical ventilation (MV) performed in respiratory failure patients to maintain lung function leads to ventilator-induced lung injury (VILI). This study investigates the role of sphingolipids and sphingolipid metabolizing enzymes in VILI using a rodent model of VILI and alveolar epithelial cells subjected to cyclic stretch (CS). MV (0 PEEP (Positive End Expiratory Pressure), 30 mL/kg, 4 h) in mice enhanced sphingosine-1-phosphate lyase (S1PL) expression, and ceramide levels, and decreased S1P levels in lung tissue, thereby leading to lung inflammation, injury and apoptosis. Accumulation of S1P in cells is a balance between its synthesis catalyzed by sphingosine kinase (SphK) 1 and 2 and catabolism mediated by S1P phosphatases and S1PL. Thus, the role of S1PL and SphK1 in VILI was investigated using Sgpl1+/- and Sphk1-/- mice. Partial genetic deletion of Sgpl1 protected mice against VILI, whereas deletion of SphK1 accentuated VILI in mice. Alveolar epithelial MLE-12 cells subjected to pathophysiological 18% cyclic stretch (CS) exhibited increased S1PL protein expression and dysregulation of sphingoid bases levels as compared to physiological 5% CS. Pre-treatment of MLE-12 cells with S1PL inhibitor, 4-deoxypyridoxine, attenuated 18% CS-induced barrier dysfunction, minimized cell apoptosis and cytokine secretion. These results suggest that inhibition of S1PL that increases S1P levels may offer protection against VILI.