sphingosine-kinase 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

Cigarette smoke (CS) is the leading cause of chronic obstructive pulmonary disease (COPD), which is characterized by chronic bronchial inflammation and emphysema. Growing evidence supports the hypothesis that dysfunctional cystic fibrosis transmembrane conductance regulator (CFTR) is critically involved in the pathogenesis of CS-mediated COPD. However, the underlying mechanism remains unclear. Here, we report that supressed CFTR expression is strongly associated with abnormal phospholipid metabolism and increased pulmonary inflammation. In a CS-exposed mouse model with COPD-like symptoms, we found that pulmonary expression of sphingosine kinase 2 (SphK2) and sphingosine-1-phosphate (S1P) secretion were significantly upregulated. Therefore, we constructed a SphK2 gene knockout (SphK2-/-) mouse. After CS exposure for six months, histological lung section staining showed disorganized alveolar structure, increased pulmonary fibrosis, and emphysema-like symptoms in wild-type (WT) mice, which were less pronounced in SphK2-/- mice. Further, SphK2 deficiency also decreased CS-induced pulmonary inflammation, which was reflected by a remarkable reduction in pulmonary infiltration of CD45+CD11b+ neutrophils subpopulation and low levels of IL-6 and IL-33 in bronchial alveolar lavage fluid. However, treatment with S1P receptor agonist suppressed CFTR expression and increased Nf-κB-p65 expression and its nuclear translocation in CS-exposed SphK2-/-mice, which also aggravated small airways fibrosis and pulmonary inflammation. In contrast, inhibition of S1P signaling with the S1P receptor analogue FTY720 rescued CFTR expression, suppressed Nf-κB-p65 expression and nuclear translocation, and alleviated pulmonary fibrosis and inflammation after CS exposure. Our results demonstrate that SphK2-mediated S1P production plays a crucial role in the pathogenesis of CS-induced COPD-like disease by impairing CFTR activity and promoting pulmonary inflammation and fibrosis.

Topics: Animals; Cigarette Smoking; Cystic Fibrosis Transmembrane Conductance Regulator; Emphysema; Inflammation; Mice; NF-kappa B; Nicotiana; Pneumonia; Pulmonary Disease, Chronic Obstructive; Pulmonary Emphysema; Pulmonary Fibrosis; Sphingosine-1-Phosphate Receptors

Infantile pneumonia is an acute inflammatory disorder of the lung caused by mycoplasma pneumonia. SPHK1 (sphingosine kinase-1) signaling pathway is involved in the process of inflammatory diseases. However, whether SphK1 regulates inflammatory responses in infantile pneumonia remains unclear. In this study, we investigated the role of SPHK1 in infantile pneumonia and its underlying mechanisms.. Serum samples of 12 patients with infantile pneumonia and healthy controls were obtained from Hunan Children's Hospital. To induce pneumonia, mice were administrated with LPS (lipopolysaccharide) into the lung. RAW264.7 cells were used as an in vitro macrophage model stimulated with LPS or PBS for 4 h.. SPHK1 mRNA level and protein level in the LPS-treated mice and patients with infantile pneumonia were significantly increased. SPHK1 promoted inflammation and lung injury in mice with infantile pneumonia. The knockdown of SPHK1 expression inhibited inflammation and restrained lung injury in mice with infantile pneumonia. SPHK1 overexpression also exacerbated inflammation in RAW264.7 cells stimulated by LPS, and SPHK1 silencing reduced inflammatory responses. We further showed that SPHK1 induced NLRP3 (NLR Family Pyrin Domain Containing 3) activity by inhibiting SIRT1 expression.. Our study demonstrated that SPHK1 promotes inflammation of infantile pneumonia by modulating NLRP3 inflammasome via the regulation of SIRT1 expression and mitochondrial permeability transition.

Topics: Animals; Inflammasomes; Inflammation; Lipopolysaccharides; Lung Injury; Mice; Mice, Inbred C57BL; NLR Family, Pyrin Domain-Containing 3 Protein; Pneumonia; Sirtuin 1

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

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

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

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

Trauma and hemorrhagic shock (T/HS) induce a systemic inflammatory response syndrome (SIRS). Neutrophils (polymorphonuclear leukocytes [PMN]) and other cells involved in acute lung injury (ALI) are activated by Ca2+ entry. Thus, inhibiting Ca2+ entry might attenuate post-traumatic lung injury. Inhibiting voltage-operated (L-type) Ca2+ channels during shock could cause cardiovascular collapse, but PMN are "nonexcitable" cells, lack L-type channels, and mobilize Ca2+ via nonspecific channels. We previously showed that PMN Ca2+ entry requires sphingosine 1-phosphate synthesis by sphingosine kinase and that both sphingosine kinase inhibition and blockade of nonspecific channels attenuate ALI when begun before shock. Pretreatment for clinical injuries, however, is impractical. Therefore, we now studied whether Ca2+ entry inhibition that begun during resuscitation from T/HS could attenuate SIRS and ALI without causing hemodynamic compromise. Male Sprague-Dawley rats underwent laparotomy and fixed-pressure shock (mean arterial pressure, 35 +/- 5 mmHg; 90 min). Sphingosine kinase inhibition or nonspecific Ca2+ channel inhibition was begun after resuscitation with 10% of shed blood. We then studied in vivo PMN activation and associated lung injury in the presence or absence of Ca2+ entry inhibition. Neither treatment worsened shock. Each treatment decreased CD11b expression, respiratory burst, PMN p38 MAP-kinase phosphorylation, PMN sequestration, and lung capillary leak in vivo. The similar results seen with two different forms of inhibition strengthen the conclusion that the biological effects seen were specific for calcium entry inhibition. Ca2+ entry inhibition is a candidate therapy for management of lung injury after shock.

Topics: Aminophenols; Animals; Calcium; Calcium Channel Blockers; Capillary Permeability; CD11b Antigen; Disease Models, Animal; Humans; Lung; Male; Neutrophils; Nitrendipine; p38 Mitogen-Activated Protein Kinases; Phosphotransferases (Alcohol Group Acceptor); Pneumonia; Rats; Rats, Sprague-Dawley; Respiratory Burst; Shock, Hemorrhagic; Shock, Traumatic; Thiazoles