sphingosine-1-phosphate and Pneumonia

Acute lung injury (ALI) attributable to sepsis or mechanical ventilation and subacute lung injury because of ionizing radiation (RILI) share profound increases in vascular permeability as a key element and a common pathway driving increased morbidity and mortality. Unfortunately, despite advances in the understanding of lung pathophysiology, specific therapies do not yet exist for the treatment of ALI or RILI, or for the alleviation of unremitting pulmonary leakage, which serves as a defining feature of the illness. A critical need exists for new mechanistic insights that can lead to novel strategies, biomarkers, and therapies to reduce lung injury. Sphingosine 1-phosphate (S1P) is a naturally occurring bioactive sphingolipid that acts extracellularly via its G protein-coupled S1P1-5 as well as intracellularly on various targets. S1P-mediated cellular responses are regulated by the synthesis of S1P, catalyzed by sphingosine kinases 1 and 2, and by the degradation of S1P mediated by lipid phosphate phosphatases, S1P phosphatases, and S1P lyase. We and others have demonstrated that S1P is a potent angiogenic factor that enhances lung endothelial cell integrity and an inhibitor of vascular permeability and alveolar flooding in preclinical animal models of ALI. In addition to S1P, S1P analogues such as 2-amino-2-(2-[4-octylphenyl]ethyl)-1,3-propanediol (FTY720), FTY720 phosphate, and FTY720 phosphonates offer therapeutic potential in murine models of lung injury. This translational review summarizes the roles of S1P, S1P analogues, S1P-metabolizing enzymes, and S1P receptors in the pathophysiology of lung injury, with particular emphasis on the development of potential novel biomarkers and S1P-based therapies for ALI and RILI.

Topics: Acute Lung Injury; Animals; Anti-Inflammatory Agents; Biomarkers; Capillary Permeability; Fingolimod Hydrochloride; Humans; Lung; Lysophospholipids; Membrane Proteins; Nerve Tissue Proteins; Phosphotransferases (Alcohol Group Acceptor); Pneumonia; Propylene Glycols; Receptors, Lysosphingolipid; Sepsis; Sphingosine; Transferases (Other Substituted Phosphate Groups); Translational Research, Biomedical

Pneumonia is one of the leading causes of death worldwide, represents a potentially life-threatening condition. In recent studies, adjuvant corticosteroids therapy has been shown to improve outcome in severe community-acquired pneumonia (CAP); however, the treatment response to corticosteroids vary. It is important to select patients likely to benefit from the treatment. Currently, the optimal patient selection of corticosteroids treatment is not yet clearly defined.. Sphingosine-1-phosphate and pneumonia (SOPN) trial is a double-blinded, randomized, placebo-controlled trial that will investigate if sphingosine-1-phosphate (S1P) can be an indicator for initiating adjuvant corticosteroids therapy in patients with severe CAP. Participants will be recruited from the emergency department and randomized to receive 20 mg of methylprednisolone twice daily or placebo for 5 days. The primary outcome will be "in-hospital mortality." Secondary outcomes will include intensive care unit (ICU) admission, length of ICU stay, length of hospital stay, and clinical outcomes at Day 7 and Day 14.. SOPN trial is the first randomized placebo-controlled trial to investigate whether S1P can be a predictive biomarker for adjuvant corticosteroids therapy in patients with severe CAP. The trial will add additional data for the appropriate use of adjuvant corticosteroids therapy in patients with severe CAP. Results from this clinical trial will provide foundational information supporting that if the S1P is appropriate for guiding the patient selection for corticosteroids adjuvant therapy.

Topics: Adjuvants, Pharmaceutic; Adult; Biomarkers; Clinical Protocols; Community-Acquired Infections; Double-Blind Method; Female; Glucocorticoids; Hospital Mortality; Humans; Intensive Care Units; Length of Stay; Lysophospholipids; Male; Methylprednisolone; Pneumonia; Sphingosine

Mechanical ventilation (MV) is a life-supporting strategy employed in the Intensive Care Unit (ICU). However, MV-associated mechanical stress exacerbates existing lung inflammation in ICU patients, resulting in limited improvement in mortality and a condition known as Ventilator-Induced Lung Injury (VILI). Sphingosine-1-phosphate (S1P) is a circulating bioactive lipid that maintains endothelial integrity primarily through S1P receptor 1 (S1PR1). During VILI, mechanical stress upregulates endothelial S1PR3 levels. Unlike S1PR1, S1PR3 mediates endothelial barrier disruption through Rho-dependent pathways. However, the specific impact of elevated S1PR3 on lung endothelial function, apart from Rho activation, remains poorly understood. In this study, we investigated the effects of S1PR3 in endothelial pathobiology during VILI using an S1PR3 overexpression adenovirus. S1PR3 overexpression caused cytoskeleton rearrangement, formation of paracellular gaps, and a modified endothelial response towards S1P. It resulted in a shift from S1PR1-dependent barrier enhancement to S1PR3-dependent barrier disruption. Moreover, S1PR3 overexpression induced an ADAM10-dependent cleavage of Vascular Endothelial (VE)-cadherin, which hindered endothelial barrier recovery. S1PR3-induced cleavage of VE-cadherin was at least partially regulated by S1PR3-mediated NFκB activation. Additionally, we employed an S1PR3 inhibitor TY-52156 in a murine model of VILI. TY-52156 effectively attenuated VILI-induced increases in bronchoalveolar lavage cell counts and protein concentration, suppressed the release of pro-inflammatory cytokines, and inhibited lung inflammation as assessed via a histological evaluation. These findings confirm that mechanical stress associated with VILI increases S1PR3 levels, thereby altering the pulmonary endothelial response towards S1P and impairing barrier recovery. Inhibiting S1PR3 is validated as an effective therapeutic strategy for VILI.

Topics: ADAM10 Protein; Amyloid Precursor Protein Secretases; Animals; Cadherins; Humans; Lysophospholipids; Membrane Proteins; Mice; Pneumonia; Receptors, Lysosphingolipid; Sphingosine; Sphingosine-1-Phosphate Receptors; Ventilator-Induced Lung Injury

Heart failure (HF) is among the main causes of death worldwide. Alterations of sphingosine-1-phosphate (S1P) signaling have been linked to HF as well as to target organ damage that is often associated with HF. S1P's availability is controlled by the cystic fibrosis transmembrane regulator (CFTR), which acts as a critical bottleneck for intracellular S1P degradation. HF induces CFTR downregulation in cells, tissues and organs, including the lung. Whether CFTR alterations during HF also affect systemic and tissue-specific S1P concentrations has not been investigated. Here, we set out to study the relationship between S1P and CFTR expression in the HF lung. Mice with HF, induced by myocardial infarction, were treated with the CFTR corrector compound C18 starting ten weeks post-myocardial infarction for two consecutive weeks. CFTR expression, S1P concentrations, and immune cell frequencies were determined in vehicle- and C18-treated HF mice and sham controls using Western blotting, flow cytometry, mass spectrometry, and qPCR. HF led to decreased pulmonary CFTR expression, which was accompanied by elevated S1P concentrations and a pro-inflammatory state in the lungs. Systemically, HF associated with higher S1P plasma levels compared to sham-operated controls and presented with higher S1P receptor 1-positive immune cells in the spleen. CFTR correction with C18 attenuated the HF-associated alterations in pulmonary CFTR expression and, hence, led to lower pulmonary S1P levels, which was accompanied by reduced lung inflammation. Collectively, these data suggest an important role for the CFTR-S1P axis in HF-mediated systemic and pulmonary inflammation.

Topics: Animals; Biomarkers; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Disease Models, Animal; Disease Susceptibility; Gene Expression; Heart Failure; Lung; Lysophospholipids; Mice; Organ Specificity; Pneumonia; Signal Transduction; Sphingosine; T-Lymphocyte Subsets

 Chronic obstructive pulmonary disease (COPD) is a significant public health concern. The patients with acute exacerbations of COPD (AECOPD) and pneumonia have similar clinical presentations. The use of conventional diagnostic markers, such as complete blood count with differential and C-reactive protein (CRP), is the current mainstream method for differentiating clinically relevant pneumonia from other mimics. However, those conventional methods have suboptimal sensitivity and specificity for patients with a clinical suspicion of infection. The limitations often cause the ambiguity of the initiation of antibiotic treatment. Recently, our pilot study suggested that the patients with pneumonia have significantly higher plasma Sphingosine-1-phosphate (S1P) levels than controls. The initial findings suggest that plasma S1P is a potential biomarker for predicting prognosis in pneumonia. The aim of this study was to evaluate the value of S1P and CRP for discriminating COPD with pneumonia and AECOPD in an Emergency Department (ED) setting..  Patients diagnosed with AECOPD or COPD with pneumonia were recruited from the Emergency Department of Wan Fang Hospital. The clinical data, demographics, and blood samples were collected upon ED admission. The concentration of plasma S1P was measured by ELISA..  Thirty-nine patients with AECOPD and 78 with COPD plus pneumonia were enrolled in this observational study. The levels of blood S1P and CRP were significantly higher in patients with COPD plus CAP compared to those in AE COPD patients. The area under the receiver operator characteristic (ROC) curve for the S1P and CRP for distinguishing between patients with COPD plus CAP and AECOPD is 0.939 (95% CI: 0.894-0.984) and 0.886 (95% CI: 0.826-0.945), whereas the combination of S1P and CRP yielded a value of 0.994 (95% CI: 0.897-1.000). By comparing with CRP or S1P, combining CRP and S1P had significantly higher AUC value for differentiating between the COPD with pneumonia group and the AECOPD group.. Our findings suggest that S1P is a potential diagnostic biomarker in distinguishing COPD with CAP from AECOPD. Additionally, the diagnostic ability of S1P can be improved when used in combination with CRP.

Topics: Aged; Biomarkers; C-Reactive Protein; Community-Acquired Infections; Diagnosis, Differential; Female; Humans; Lysophospholipids; Male; Pneumonia; Prospective Studies; Pulmonary Disease, Chronic Obstructive; Sphingosine

Sphingosine-1-phosphate (S1P) is involved in inflammatory signaling/s associated with the development of respiratory disorders, including cancer. However, the underlying mechanism/s are still elusive. The aim of this study was to investigate the role of S1P on circulating blood cells obtained from healthy volunteers and non-small cell lung cancer (NSCLC) patients. To pursue our goal, peripheral blood mononuclear cells (PBMCs) were isolated and stimulated with S1P. We found that the administration of S1P did not induce healthy PBMCs to release pro-inflammatory cytokines. In sharp contrast, S1P significantly increased the levels of TNF-α and IL-6 from lung cancer-derived PBMCs. This effect was S1P receptor 3 (S1PR3)-dependent. The pharmacological blockade of ceramidase and sphingosine kinases (SPHKs), key enzymes for S1P synthesis, completely reduced the release of both TNF-α and IL-6 after S1P addition on lung cancer-derived PBMCs. Interestingly, S1P-induced IL-6, but not TNF-α, release from lung cancer-derived PBMCs was mTOR- and K-Ras-dependent, while NF-κB was not involved. These data identify S1P as a bioactive lipid mediator in a chronic inflammation-driven diseases such as NSCLC. In particular, the higher presence of S1P could orchestrate the cytokine milieu in NSCLC, highlighting S1P as a pro-tumor driver.

Topics: Carcinoma, Non-Small-Cell Lung; Cytokines; Humans; Inflammation; Interleukin-6; Leukocytes, Mononuclear; Lung Neoplasms; Lysophospholipids; Pneumonia; Sphingosine; Tumor Necrosis Factor-alpha

Acute ethanol intoxication increases the risk of sepsis and aggravates the symptoms of sepsis and lung injury. Therefore, this study aimed to explore whether sphingosine kinase 1 (SphK1)/sphingosine-1-phosphate (S1P)/S1P receptor 1 (S1PR1) signaling pathway functions in lung injury caused by acute ethanol intoxication-enhanced sepsis, as well as its underlying mechanism. The acute ethanol intoxication model was simulated by intraperitoneally administering mice with 32% ethanol solution, and cecal ligation and puncture (CLP) was used to construct the sepsis model. The lung tissue damage was observed by hematoxylin-eosin (H&E) staining, and the wet-to-dry (W/D) ratio was used to evaluate the degree of pulmonary edema. Inflammatory cell counting and protein concentration in bronchoalveolar lavage fluid (BALF) were, respectively, detected by hemocytometer and bicinchoninic acid (BCA) method. The levels of tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-1β, and IL-18 in BALF were detected by their commercial enzyme-linked immunosorbent assay (ELISA) kits. The myeloperoxidase (MPO) activity and expression of apoptosis-related proteins and SphK1/S1P/S1PR1 pathway-related proteins were, respectively, analyzed by MPO ELISA kit and Western blot analysis. The cell apoptosis in lung tissues was observed by TUNEL assay. Acute ethanol intoxication (EtOH) decreased the survival rate of mice and exacerbated the lung injury caused by sepsis through increasing pulmonary vascular permeability, neutrophil infiltration, release of inflammatory factors, and cell apoptosis. In addition, EtOH could activate the SphK1/S1P/S1PR1 pathway in CLP mice. However, PF-543, as a specific inhibitor of SphK1, could partially reverse the deleterious effects on lung injury of CLP mice. PF-543 alleviated lung injury caused by sepsis in acute ethanol intoxication rats by suppressing the SphK1/S1P/S1PR1 signaling pathway.

Topics: Alcoholic Intoxication; Animals; Apoptosis; Cytokines; Disease Models, Animal; Enzyme Inhibitors; Inflammation Mediators; Lung; Lung Injury; Lysophospholipids; Male; Methanol; Mice, Inbred C57BL; Neutrophil Infiltration; Oxidative Stress; Phosphotransferases (Alcohol Group Acceptor); Pneumonia; Pulmonary Edema; Pyrrolidines; Sepsis; Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors; Sulfones

Sphingosine-1-phosphate (S1P) is involved in the regulation of important cellular processes, including immune-cell trafficking and proliferation. Altered S1P signaling is strongly associated with inflammation, cancer progression, and atherosclerosis; however, the mechanisms underlying its pathophysiologic effects are only partially understood. This study evaluated the effects of S1P in vitro and in vivo on the biosynthesis of leukotrienes (LTs), which form a class of lipid mediators involved in the pathogenesis of inflammatory diseases. Here, we report for the first time that S1P potently suppresses LT biosynthesis in Ca

Topics: Animals; Anti-Inflammatory Agents; Arachidonate 5-Lipoxygenase; Arachidonic Acid; Calcium; Cell Line; Female; Humans; Lysophospholipids; Mice; Mice, Inbred C57BL; Neutrophils; Pneumonia; Reactive Oxygen Species; Receptors, Lysosphingolipid; Signal Transduction; Sphingosine; Substrate Specificity

Early determination of the severity of Community-Acquired Pneumonia (CAP) is essential for better disease prognosis. Current predictors are suboptimal, and their clinical utility remains to be defined, highlighting the need for developing biomarkers with efficacious prognostic value. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid with a documented regulatory role in immune defense and maintenance of endothelial barrier integrity. For early diagnose of CAP and recognition of severe CAP patients, we conduct this pilot study to access the potential utility of the circulating S1P in an Emergency department setting. In the prospective study, plasma S1P levels were quantified in healthy controls and patients with CAP. Also, their discriminating power was assessed by receiver operating characteristic analysis. The association between S1P levels and disease severity indices was assessed by Spearman correlation and logistic regression tests. Patients with CAP had significantly higher plasma S1P levels than healthy individuals (CAP: 27.54 ng/ml, IQR = 14.37-49.99 ng/ml; Controls: 10.58 ng/ml, IQR = 4.781-18.91 ng/ml; p < 0.0001). S1P levels were inversely correlated with disease severity in patients with CAP. Based on multivariate logistic regression analysis, the plasma S1P concentrations showed significant predicting power for mortality (OR: 0.909; CI: 0.801-0.985; p < 0.05), intensive care unit admission (OR: 0.89; CI: 0.812-0.953; p < 0.005) and long hospital stay (OR: 0.978; CI: 0.961-0.992; p < 0.005). Interestingly, significantly elevated levels of S1P were noted in patients who received methylprednisolone treatment during hospitalization. These results suggest that S1P may be associated with the pathogenesis of CAP and may have prognostic utility in CAP and its therapy, especially in the Emergency Department setting.

Topics: Aged; Biomarkers; Community-Acquired Infections; Emergency Service, Hospital; Female; Hospitalization; Humans; Intensive Care Units; Logistic Models; Lysophospholipids; Male; Middle Aged; Pilot Projects; Pneumonia; Prognosis; Sphingosine

Topics: Aldehyde-Lyases; Animals; Biological Transport; Body Weight; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Cytokines; Dendritic Cells; Disease Models, Animal; Enzyme Inhibitors; Epithelial Cells; Imidazoles; Lipopolysaccharides; Lung; Lysophospholipids; Mice, Inbred C57BL; Mucin 5AC; Mutation; Myeloid Cells; N-Formylmethionine Leucyl-Phenylalanine; Oximes; Pneumonia; Salivary Glands; Sphingosine; X-Ray Microtomography

Compelling evidence suggests the involvement of sphingosine-1-phosphate (S1P) in the pathogenesis of asthma. The systemic administration of S1P causes asthma like features in the mouse involving mast cells. In this study we investigated whether disodium cromoglycate (DSCG), administered as a preventative treatment as in human therapy, could affect S1P effects on airways. BALB/c mice, treated with DSCG, received subcutaneous administration of S1P. Bronchi and pulmonary tissues were collected and functional, molecular and cellular studies were performed. DSCG inhibited S1P-induced airway hyper-reactivity as well as pulmonary inflammation. DSCG decreased the recruitment of solely mast cells and B cells in the lung. IgE serum levels, prostaglandin D

Topics: Administration, Cutaneous; Animals; Asthma; B-Lymphocytes; Bronchi; Cromolyn Sodium; Female; Humans; Immunoglobulin E; Lysophospholipids; Mast Cells; Mice; Mice, Inbred BALB C; Pneumonia; Receptors, IgE; Sphingosine; T-Lymphocytes

Sphingosine-1-phosphate (S1P) has been widely associated with inflammation-based lung pathologies. Because B cells play a critical role as antigen-presenting and/or Ig-producing cells during asthmatic conditions, we wanted to dissect the role of these cells in S1P-dependent airway hyperreactivity and inflammation. Mice were sensitized to ovalbumin or exposed to S1P. Ovalbumin sensitization caused airway hyperreactivity coupled to an increased lung infiltration of B cells, which was significantly reduced after the inhibition of sphingosine kinases I/II. Similarly, the sole administration of S1P increased bronchial reactivity compared with vehicle and was accompanied by a higher influx of B cells in a time-dependent manner. This effect was associated with higher levels of IL-13, transforming growth factor-β, IL-10, and T regulatory cells. In addition, isolated S1P-derived lung B cells increased CD4(+) and CD8(+) T cell proliferation in vitro, and their suppressive nature at Day 14 was associated with the higher release of transforming growth factor-β and IL-10 when they were cocultured. Therefore, to prove the role of B cells in S1P-mediated airway inflammation, and because CD20 expression, contrary to major hystocompatibility complex I and major hystocompatibility complex II, was up-regulated at Day 14, CD20(+) B cells were depleted by means of a specific monoclonal antibody. The absence of CD20(+) B cells increased airway reactivity and inflammation in S1P-treated mice compared with control mice. These data imply that sphingosine kinase/S1P-mediated airway inflammation is countered by B cells via the induction of an immune-suppressive environment to reduce asthma-like outcomes in mice.

Topics: Animals; Antibodies, Monoclonal; Antigens, CD20; B-Lymphocytes; Bronchial Hyperreactivity; Bronchoconstriction; Cell Proliferation; Chemotaxis, Leukocyte; Disease Models, Animal; Female; Inflammation Mediators; Interleukin-10; Interleukin-13; Lung; Lymphocyte Activation; Lysophospholipids; Mice, Inbred BALB C; Ovalbumin; Phosphotransferases (Alcohol Group Acceptor); Pneumonia; Protein Kinase Inhibitors; Sphingosine; T-Lymphocytes, Regulatory; Time Factors; Transforming Growth Factor beta

Sphingosine-1-phosphate (S1P) has been shown to be involved in the asthmatic disease as well in preclinical mouse experimental models of this disease. The aim of this study was to understand the mechanism(s) underlying S1P effects on the lung.. BALB/c, mast cell-deficient and Nude mice were injected with S1P (s.c.) on days 0 and 7. Functional, molecular and cellular studies were performed.. S1P administration to BALB/c mice increased airway smooth muscle reactivity, mucus production, PGD2 , IgE, IL-4 and IL-13 release. These features were associated to a higher recruitment of mast cells to the lung. Mast cell-deficient Kit (W) (-sh/) (W) (-sh) mice injected with S1P did not display airway smooth muscle hyper-reactivity. However, lung inflammation and IgE production were still present. Treatment in vivo with the anti-CD23 antibody B3B4, which blocks IgE production, inhibited both S1P-induced airway smooth muscle reactivity in vitro and lung inflammation. S1P administration to Nude mice did not elicit airway smooth muscle hyper-reactivity and lung inflammation. Naïve (untreated) mice subjected to the adoptive transfer of CD4+ T-cells harvested from S1P-treated mice presented all the features elicited by S1P in the lung.. S1P triggers a cascade of events that sequentially involves T-cells, IgE and mast cells reproducing several asthma-like features. This model may represent a useful tool for defining the role of S1P in the mechanism of action of currently-used drugs as well as in the development of new therapeutic approaches for asthma-like diseases.

Topics: Animals; Bronchial Hyperreactivity; CD4-Positive T-Lymphocytes; Immunoglobulin E; Interleukin-13; Interleukin-4; Lysophospholipids; Mast Cells; Mice, Inbred BALB C; Mice, Knockout; Mice, Nude; Pneumonia; Prostaglandin D2; Sphingosine

The increased use of inhaled nicotine via e-cigarettes has unknown risks to lung health. Having previously shown that cigarette smoke (CS) extract disrupts the lung microvasculature barrier function by endothelial cell activation and cytoskeletal rearrangement, we investigated the contribution of nicotine in CS or e-cigarettes (e-Cig) to lung endothelial injury. Primary lung microvascular endothelial cells were exposed to nicotine, e-Cig solution, or condensed e-Cig vapor (1-20 mM nicotine) or to nicotine-free CS extract or e-Cig solutions. Compared with nicotine-containing extract, nicotine free-CS extract (10-20%) caused significantly less endothelial permeability as measured with electric cell-substrate impedance sensing. Nicotine exposures triggered dose-dependent loss of endothelial barrier in cultured cell monolayers and rapidly increased lung inflammation and oxidative stress in mice. The endothelial barrier disruptive effects were associated with increased intracellular ceramides, p38 MAPK activation, and myosin light chain (MLC) phosphorylation, and was critically mediated by Rho-activated kinase via inhibition of MLC-phosphatase unit MYPT1. Although nicotine at sufficient concentrations to cause endothelial barrier loss did not trigger cell necrosis, it markedly inhibited cell proliferation. Augmentation of sphingosine-1-phosphate (S1P) signaling via S1P1 improved both endothelial cell proliferation and barrier function during nicotine exposures. Nicotine-independent effects of e-Cig solutions were noted, which may be attributable to acrolein, detected along with propylene glycol, glycerol, and nicotine by NMR, mass spectrometry, and gas chromatography, in both e-Cig solutions and vapor. These results suggest that soluble components of e-Cig, including nicotine, cause dose-dependent loss of lung endothelial barrier function, which is associated with oxidative stress and brisk inflammation.

Topics: Animals; Capillary Permeability; Cell Proliferation; Cells, Cultured; Ceramides; Electric Impedance; Electronic Nicotine Delivery Systems; Endothelium, Vascular; Female; Gas Chromatography-Mass Spectrometry; Humans; Immunoblotting; Lysophospholipids; Mice; Mice, Inbred C57BL; Nicotine; Nicotinic Agonists; Oxidative Stress; Phosphorylation; Pneumonia; Rats; Signal Transduction; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Sphingosine

Bronchopulmonary dysplasia of the premature newborn is characterized by lung injury, resulting in alveolar simplification and reduced pulmonary function. Exposure of neonatal mice to hyperoxia enhanced sphingosine-1-phosphate (S1P) levels in lung tissues; however, the role of increased S1P in the pathobiological characteristics of bronchopulmonary dysplasia has not been investigated. We hypothesized that an altered S1P signaling axis, in part, is responsible for neonatal lung injury leading to bronchopulmonary dysplasia. To validate this hypothesis, newborn wild-type, sphingosine kinase1(-/-) (Sphk1(-/-)), sphingosine kinase 2(-/-) (Sphk2(-/-)), and S1P lyase(+/-) (Sgpl1(+/-)) mice were exposed to hyperoxia (75%) from postnatal day 1 to 7. Sphk1(-/-), but not Sphk2(-/-) or Sgpl1(+/-), mice offered protection against hyperoxia-induced lung injury, with improved alveolarization and alveolar integrity compared with wild type. Furthermore, SphK1 deficiency attenuated hyperoxia-induced accumulation of IL-6 in bronchoalveolar lavage fluids and NADPH oxidase (NOX) 2 and NOX4 protein expression in lung tissue. In vitro experiments using human lung microvascular endothelial cells showed that exogenous S1P stimulated intracellular reactive oxygen species (ROS) generation, whereas SphK1 siRNA, or inhibitor against SphK1, attenuated hyperoxia-induced S1P generation. Knockdown of NOX2 and NOX4, using specific siRNA, reduced both basal and S1P-induced ROS formation. These results suggest an important role for SphK1-mediated S1P signaling-regulated ROS in the development of hyperoxia-induced lung injury in a murine neonatal model of bronchopulmonary dysplasia.

Topics: Aldehyde-Lyases; Animals; Animals, Newborn; Bronchopulmonary Dysplasia; Disease Models, Animal; Down-Regulation; Endothelial Cells; Humans; Hyperoxia; Lysophospholipids; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; NADPH Oxidase 2; NADPH Oxidase 4; NADPH Oxidases; Phosphotransferases (Alcohol Group Acceptor); Pneumonia; Pulmonary Alveoli; rac1 GTP-Binding Protein; Reactive Oxygen Species; Signal Transduction; Sphingosine

The complement anaphylatoxin C5a has a pathogenetic role in endotoxin-induced lung inflammatory injury by regulating phagocytic cell migration and activation. Endotoxin and C5a activate the enzyme sphingosine kinase (Sphk) 1 to generate the signaling lipid sphingosine-1-phosphate (S1P), a critical regulator of phagocyte function. We assessed the function of Sphk1 and S1P in experimental lung inflammatory injury and determined their roles in anaphylatoxin receptor signaling and on the expression of the two C5a receptors, C5aR (CD88) and C5L2, on phagocytes. We report that Sphk1 gene deficient (Sphk1(-/-)) mice had augmented lung inflammatory response to endotoxin compared to wild type mice. Sphk1 was required for C5a-mediated reduction in cytokine and chemokine production by macrophages. Moreover, neutrophils from Sphk1(-/-) mice failed to upregulate the anaphylatoxin receptor C5L2 in response to LPS. Exogenous S1P restored C5L2 cell surface expression of Sphk1(-/-) mouse neutrophils to wild type levels but had no effect on cell surface expression of the other anaphylatoxin receptor, CD88. These results provide the first genetic evidence of the crucial role of Sphk1 in regulating the balance between expression of CD88 and C5L2 in phagocytes. S1P-mediated up-regulation of C5L2 is a novel therapeutic target for mitigating endotoxin-induced lung inflammatory injury.

Topics: Anaphylatoxins; Animals; Bone Marrow; Cytokines; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Lipopolysaccharides; Lysophospholipids; Macrophages; Mice; Mice, Inbred C57BL; Mice, Knockout; Neutrophils; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Pneumonia; Receptor, Anaphylatoxin C5a; Receptors, Chemokine; Sepsis; Signal Transduction; Sphingosine

We recently reported that integrin α(v)β(3) is necessary for vascular barrier protection in mouse models of acute lung injury and peritonitis. Here, we used mass spectrometric sequencing of integrin complexes to isolate the novel β(3)-integrin binding partner IQGAP1. Like integrin β(3), IQGAP1 localized to the endothelial cell-cell junction after sphingosine-1-phosphate (S1P) treatment, and IQGAP1 knockdown prevented cortical actin formation and barrier enhancement in response to S1P. Furthermore, knockdown of IQGAP1 prevented localization of integrin α(v)β(3) to the cell-cell junction. Similar to β(3)-null animals, IQGAP1-null mice had increased pulmonary vascular leak compared with wild-type controls 3 days after intratracheal LPS. In an Escherichia coli pneumonia model, IQGAP1 knockout mice had increased lung weights, lung water, and lung extravascular plasma equivalents of (125)I-labeled albumin compared with wild-type controls. Taken together, these experiments indicate that IQGAP1 is necessary for S1P-mediated vascular barrier protection during acute lung injury and is required for junctional localization of the barrier-protective integrin α(v)β(3).

Topics: Actins; Acute Lung Injury; Animals; Cells, Cultured; Endothelial Cells; Endothelium, Vascular; Integrin alphaVbeta3; Lipopolysaccharides; Lung; Lysophospholipids; Mice; Mice, Knockout; Pneumonia; Protein Binding; ras GTPase-Activating Proteins; Sphingosine

A defining feature of acute lung injury (ALI) is the increased lung vascular permeability and alveolar flooding, which leads to associated morbidity and mortality. Specific therapies to alleviate the unremitting vascular leak in ALI are not currently clinically available; however, our prior studies indicate a protective role for sphingosine-1-phosphate (S1P) in animal models of ALI with reductions in lung edema. As S1P levels are tightly regulated by synthesis and degradation, we tested the hypothesis that inhibition of S1P lyase (S1PL), the enzyme that irreversibly degrades S1P via cleavage, could ameliorate ALI. Intratracheal instillation of LPS to mice enhanced S1PL expression, decreased S1P levels in lung tissue, and induced lung inflammation and injury. LPS challenge of wild-type mice receiving 2-acetyl-4(5)-[1(R),2(S),3(R),4-tetrahydroxybutyl]-imidazole to inhibit S1PL or S1PL(+/-) mice resulted in increased S1P levels in lung tissue and bronchoalveolar lavage fluids and reduced lung injury and inflammation. Moreover, down-regulation of S1PL expression by short interfering RNA (siRNA) in primary human lung microvascular endothelial cells increased S1P levels, and attenuated LPS-mediated phosphorylation of p38 mitogen-activated protein kinase and I-κB, IL-6 secretion, and endothelial barrier disruption via Rac1 activation. These results identify a novel role for intracellularly generated S1P in protection against ALI and suggest S1PL as a potential therapeutic target.

Topics: Acute Lung Injury; Aldehyde-Lyases; Animals; Bronchoalveolar Lavage; Cells, Cultured; Endothelium, Vascular; Humans; Immunoblotting; Injections, Intraperitoneal; Interleukin-6; Lipopolysaccharides; Lysophospholipids; Mice; Mice, Inbred C57BL; Mice, Knockout; NF-kappa B; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Pneumonia; RNA, Small Interfering; Sphingosine; Tandem Mass Spectrometry

Sphingosine 1-phosphate (S1P) is a key endogenous regulator of the response to lung injury, maintaining endothelial barrier integrity through interaction with one of its receptors, S1P(1). The short-term administration of S1P or S1P(1) receptor agonists enhances endothelial monolayer barrier function in vitro, and attenuates injury-induced vascular leak in the lung and other organ systems in vivo. Although S1P(1) agonists bind to and activate S1P(1), several of these agents also induce receptor internalization and degradation, and may therefore act as functional antagonists of S1P(1) after extended exposure. Here we report on the effects of prolonged exposure to these agents in bleomycin-induced lung injury. We demonstrate that repeated administration of S1P(1) agonists dramatically worsened lung injury after bleomycin challenge, as manifested by increased vascular leak and mortality. Consistent with these results, prolonged exposure to S1P(1) agonists in vitro eliminated the ability of endothelial cell monolayers to respond appropriately to the barrier-protective effects of S1P, indicating a loss of normal S1P-S1P(1) signaling. As bleomycin-induced lung injury progressed, continued exposure to S1P(1) agonists also resulted in increased pulmonary fibrosis. These data indicate that S1P(1) agonists can act as functional antagonists of S1P(1) on endothelial cells in vivo, which should be considered in developing these agents as therapies for vascular leak syndromes. Our findings also support the hypothesis that vascular leak is an important component of the fibrogenic response to lung injury, and suggest that targeting the S1P-S1P(1) pathway may also be an effective therapeutic strategy for fibrotic lung diseases.

Topics: Animals; beta-Alanine; Bleomycin; Blood Coagulation; Endothelial Cells; Fibrosis; Fingolimod Hydrochloride; Humans; Lung Injury; Lysophospholipids; Male; Mice; Mice, Inbred C57BL; Oxadiazoles; Pneumonia; Propylene Glycols; Pulmonary Alveoli; Receptors, Lysosphingolipid; Signal Transduction; Sphingosine; Survival Analysis; Thiophenes; Vascular Diseases

The mechanism underlying the protective effect of sphingosine kinase 1 (SphK1) in inflammatory injury is not clear. We demonstrated using SphK1-null mice (SphK1(-/-)) the crucial role of SphK1 in suppressing lipopolysaccharide-induced neutrophil oxidant production and sequestration in lungs and mitigating lung inflammatory injury. This effect of SphK1 was independent of the production of sphingosine 1-phosphate, the product of SphK1 activity. The anti-inflammatory effect of SphK1 in the lipopolysaccharide model was mediated through SphK1 interaction with JNK. SphK1 stabilization of JNK in turn inhibited JNK binding to the JNK-interacting protein 3 (JIP3) and thus abrogated the activation of NADPH oxidase and oxidant generation and resultant NF-kappaB activation. Therefore, SphK1-mediated down-regulation of JNK activity serves to dampen inflammation and tissue injury.

Topics: Adaptor Proteins, Signal Transducing; Animals; Down-Regulation; Enzyme Activation; Lipopolysaccharides; Lung; Lysophospholipids; MAP Kinase Kinase 4; Mice; Mice, Knockout; NADPH Oxidases; Nerve Tissue Proteins; Neutrophils; Oxidants; Phosphotransferases (Alcohol Group Acceptor); Pneumonia; Sphingosine

The sphingomyelin/ceramide signaling pathway is an important component of many cellular processes implicated in the pathogenesis of lung disease. Acid sphingomyelinase (ASM) is a key mediator of this pathway, but its specific role in pulmonary fibrosis has not been previously investigated. Here we used the bleomycin model of pulmonary fibrosis to investigate fibrotic responses in normal and ASM knockout (ASM(-/-)) mice, and in NIH3T3 fibroblasts with and without ASM siRNA treatment.. Mice and cells with and without ASM activity were treated with bleomycin, and the effects on lung inflammation, formation of collagen producing myofibroblasts, and apoptosis were assessed.. The development of bleomycin-induced inflammation and fibrosis in wildtype mice correlated with the rapid activation of ASM, and was markedly attenuated in the absence of ASM activity. Along with the elevated ASM activity, there also was an elevation of acid ceramidase (AC) activity, which was sustained for up to 14 days post-bleomycin treatment. Studies in NIH3T3 fibroblasts confirmed these findings, and revealed a direct effect of ASM/AC activation on the formation of myofibroblasts. Cell studies also showed that a downstream effect of bleomycin treatment was the production of sphingosine-1-phosphate.. These data demonstrate that the sphingomyelin/ceramide signaling pathway is involved in the pathogenesis of bleomycin-induced pulmonary fibrosis, and suggest that inhibition of ASM may potentially slow the fibrotic process in the lung.

Topics: Actins; Animals; Antibiotics, Antineoplastic; Bleomycin; Lysophospholipids; Mice; Mice, Inbred C57BL; Mice, Knockout; NIH 3T3 Cells; Pneumonia; Pulmonary Fibrosis; RNA Interference; RNA, Small Interfering; Signal Transduction; Sphingomyelin Phosphodiesterase; Sphingosine

Two mammalian sphingosine kinase (SphK) isoforms, SphK1 and SphK2, possess identical kinase domains but have distinct kinetic properties and subcellular localizations, suggesting each has one or more specific roles in sphingosine-1-phosphate (S1P) generation. Although both kinases use sphingosine as a substrate to generate S1P, the mechanisms controlling SphK activation and subsequent S1P generation during lung injury are not fully understood. In this study, we established a murine lung injury model to investigate LPS-induced lung injury in SphK1 knockout (SphK1(-/-)) and wild-type (WT) mice. We found that SphK1(-/-) mice were much more susceptible to LPS-induced lung injury compared with their WT counterparts, quantified by multiple parameters including cytokine induction. Intriguingly, overexpression of WT SphK1 delivered by adenoviral vector to the lungs protected SphK1(-/-) mice from lung injury and attenuated the severity of the response to LPS. However, adenoviral overexpression of a SphK1 kinase-dead mutant (SphKKD) in SphK1(-/-) mouse lungs further exacerbated the response to LPS as well as the extent of lung injury. WT SphK2 adenoviral overexpression also failed to provide protection and, in fact, augmented the degree of LPS-induced lung injury. This suggested that, in vascular injury, S1P generated by SphK2 activation plays a distinctly separate role compared with SphK1-dependent S1P generation and survival signaling. Microarray and real-time RT-PCR analysis of SphK1 and SphK2 expression levels during lung injury revealed that, in WT mice, LPS treatment caused significantly enhanced SphK1 expression ( approximately 5x) levels within 6 h, which declined back to baseline levels by 24 h posttreatment. In contrast, expression of SphK2 was gradually induced following LPS treatment and was elevated within 24 h. Collectively, our results for the first time demonstrate distinct functional roles of the two SphK isoforms in the regulation of LPS-induced lung injury.

Topics: Adenoviridae; Animals; Gene Deletion; Gene Expression Regulation, Enzymologic; Gene Transfer Techniques; Lipopolysaccharides; Lung; Lung Injury; Lysophospholipids; Membrane Proteins; Mice; Mice, Inbred C57BL; Phosphoric Monoester Hydrolases; Phosphotransferases (Alcohol Group Acceptor); Pneumonia; Pulmonary Edema; Sphingosine; Time Factors; Tumor Necrosis Factor-alpha

The phospholipids sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) act via transmembrane receptors S1P 1-5 and LPA 1-3, respectively. Both have been implicated in inflammatory responses. S1P and LPA receptor profiles on neutrophils of patients with pneumonia compared with healthy subjects were determined by PCR and Western blotting. Chemotaxis studies were performed to assess functional differences. S1P or LPA receptors were immunoprecipitated from neutrophils to assess receptor heterodimerization with CXCR1, an IL-8 receptor, by Western blotting. Receptors S1P 1, 4, and 5 and LPA 2 were expressed on neutrophils from both subject groups, but S1P 3 and LPA 1 receptor expression was mainly confined to neutrophils of patients with pneumonia. Chemotaxis of neutrophils from patients with pneumonia compared with control subjects was significantly increased in response to S1P and LPA. Pretreatment with S1P or LPA reduced IL-8-induced neutrophil chemotaxis and transcriptional expression of the CXCR1 receptor. Receptors S1P 3 and 4 and LPA 1 formed constitutive heterodimers with CXCR1. LPA treatment reduced the amount of LPA 1/CXCR1 heterodimer. Therefore, profiles of S1P and LPA receptors differ between neutrophils of patients with pneumonia and control subjects, with consequences for neutrophil function.

Topics: Adult; Aged; Aged, 80 and over; Case-Control Studies; Chemotaxis; Dimerization; Female; Gene Expression; Humans; Lysophospholipids; Male; Middle Aged; Neutrophils; Pneumonia; Protein Isoforms; Receptors, Interleukin-8A; Receptors, Lysophosphatidic Acid; Receptors, Lysophospholipid; Reference Values; Sphingosine

Our prior in vitro studies indicate that sphingosine 1-phosphate (S1P), a phospholipid angiogenic factor, produces endothelial cell barrier enhancement through ligation of endothelial differentiation gene family receptors. We hypothesized that S1P may reduce the vascular leak associated with acute lung injury and found that S1P infusion produced a rapid and significant reduction in lung weight gain (more than 50%) in the isolated perfused murine lung. The effect of S1P was next assessed in a murine model of LPS-mediated microvascular permeability and inflammation with marked increases in parameters of lung injury at both 6 and 24 hours after intratracheal LPS. Each parameter assessed was significantly reduced by intravenous S1P (1 microM final) and in selected experiments by the S1P analogue FTY720 (0.1 mg/kg, intraperitoneally) delivered 1 hour after LPS. S1P produced an approximately 40-50% reduction in LPS-mediated extravasation of Evans blue dye albumin, bronchoalveolar lavage protein content, and lung tissue myeloperoxidase activity (reflecting phagocyte infiltration). Consistent with systemic barrier enhancement, S1P significantly decreased Evans blue dye albumin extravasation and myeloperoxidase content in renal tissues of LPS-treated mice. These studies indicate that S1P significantly decreases pulmonary/renal vascular leakage and inflammation in a murine model of LPS-mediated acute lung injury and may represent a novel therapeutic strategy for vascular barrier dysfunction.

Topics: Animals; Bronchoalveolar Lavage Fluid; Capillary Permeability; Disease Models, Animal; Endothelial Cells; Endotoxins; Fingolimod Hydrochloride; Immunosuppressive Agents; Kidney; Kidney Diseases; Lipopolysaccharides; Lung; Lysophospholipids; Male; Mice; Mice, Inbred C57BL; Neutrophils; Organ Size; Perfusion; Peroxidase; Pneumonia; Propylene Glycols; Respiratory Distress Syndrome; Sphingosine; Time Factors