sphingosine-kinase and Sepsis

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

Previously, we confirmed that sphingosine kinase 1 (SphK1) inhibition improves sepsis-associated liver injury. High-mobility group box 1 (HMGB1) translocation participates in the development of acute liver failure. However, little information is available on the association between SphK1 and HMGB1 translocation during sepsis-associated liver injury. In the present study, we aimed to explore the effect of SphK1 inhibition on HMGB1 translocation and the underlying mechanism during sepsis-associated liver injury. Primary Kupffer cells and hepatocytes were isolated from SD rats. The rat model of sepsis-associated liver damage was induced by intraperitoneal injection with lipopolysaccharide (LPS). We confirmed that Kupffer cells were the cells primarily secreting HMGB1 in the liver after LPS stimulation. LPS-mediated HMGB1 expression, intracellular translocation, and acetylation were dramatically decreased by SphK1 inhibition. Nuclear histone deacetyltransferase 4 (HDAC4) translocation and E1A-associated protein p300 (p300) expression regulating the acetylation of HMGB1 were also suppressed by SphK1 inhibition. HDAC4 intracellular translocation has been reported to be controlled by the phosphorylation of HDAC4. The phosphorylation of HDAC4 is modulated by CaMKII-δ. However, these changes were completely blocked by SphK1 inhibition. Additionally, by performing coimmunoprecipitation and pull-down assays, we revealed that SphK1 can directly interact with CaMKII-δ. The colocalization of SphK1 and CaMKII-δ was verified in human liver tissues with sepsis-associated liver injury. In conclusion, SphK1 inhibition diminishes HMGB1 intracellular translocation in sepsis-associated liver injury. The mechanism is associated with the direct interaction of SphK1 and CaMKII-δ.

Topics: Acetylation; Animals; Calcium-Calmodulin-Dependent Protein Kinase Type 2; E1A-Associated p300 Protein; Histone Deacetylases; HMGB1 Protein; Humans; Kupffer Cells; Liver; Male; Mice; Models, Biological; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Protein Binding; Protein Transport; Rats, Sprague-Dawley; RAW 264.7 Cells; Repressor Proteins; Sepsis

Sepsis mouse models revealed thymus atrophy, characterised by decreased thymus weight and loss of thymocytes due to apoptosis. Mice suffered from lymphopenia, a lack of T cells in the periphery, which attenuates their ability to fight against recurring and secondary infections during sepsis progression. Key players in thymus atrophy are IL-6, which is directly involved in thymus involution, and the sphingosine-1-phosphate - sphingosine-1-phosphate receptor 1 signaling, influencing thymocytes emigration. In healthy individuals a sphingosine-1-phosphate (S1P) gradient from lymphoid organs to the circulatory system serves as signal for mature T cell egress. In the present study we investigated, whether inhibition of S1P generation improves thymus involution. In sepsis, induced by cecal ligation and puncture (CLP), S1P in the thymus increased, while it decreased in serum, thus disrupting the naturally occurring S1P gradient. As a potential source of S1P we identified increased numbers of apoptotic cells in the thymic cortex of septic mice. Pharmacological inhibition of the S1P generating sphingosine kinases, by 4- [[4-(4-Chlorophenyl)-2-thiazolyl]amino]phenol (SK I-II), administered directly following CLP, prevented thymus atrophy. This was reflected by lymphocytosis, diminished apoptosis, decreased IL-6 expression, and an unaltered thymus weight. In addition SK I-II-treatment preserved the S1P balance and prevented S1P-dependent internalization of the sphingosine-1-phosphate receptor 1. Our data suggest that inhibition of sphingosine kinase and thus, S1P generation during sepsis restores thymic T cell egress, which might improve septic outcome.

Topics: Aminophenols; Animals; Apoptosis; Atrophy; Cecum; Disease Models, Animal; Interleukin-6; Lymphocytosis; Lymphopenia; Lysophospholipids; Male; Mice; Mice, Inbred C57BL; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; Sepsis; Sphingosine; Thiazoles; Thymocytes; Thymus Gland

The sphingosine kinase 1 (SphK1)/sphingosine-1-phosphate (S1P) pathway plays a key role in inflammation. Parenteral nutrition containing n-3 polyunsaturated fatty acids (n-3 PUFA) may regulate inflammatory reactions. The aim of this study is to determine whether n-3 PUFA may improve inflammatory responses by neutralizing SphK1 signaling. Rat models of parenteral nutrition, cecal ligation and puncture (CLP)-induced sepsis were generated. Male Sprague-Dawley rats were operated for CLP on day 2 after venous catheterization. The rats were randomized to receive normal saline (NS; n = 20), parenteral nutrition (PN; n = 20), or PN + fish oil (FO; n = 20) for 5 days. The daily intake of fish oil (1.25-2.82 g EPA and 1.44-3.09 g DHA per 100 ml) in the FO group was approximately 1.8 g/kg body weight/day. Rats in the control group (n = 10) were subjected to sham operation and received a chow diet. Spleen tissues were collected for SphK1 and S1P receptor expression analysis. Our data showed that n-3 PUFA ameliorated the survival rate. SphK1 expression and its enzymatic activity were significantly upregulated in sepsis rats. Furthermore, mRNA and protein levels of S1PR3, but not S1PR1, were also facilitated after CLP. However, PN + FO dramatically decreased SphK1 mRNA level and its enzymatic activity. S1PR3 expression was also attenuated by FO addition. In conclusion, the anti-inflammatory effect of n-3 PUFA may be linked to the inhibition of the SphK1/S1P pathway in a rat model of parenteral nutrition and CLP-induced sepsis.

Topics: Animals; Disease Models, Animal; Fatty Acids, Omega-3; Inflammation; Male; Parenteral Nutrition; Phosphotransferases (Alcohol Group Acceptor); Protein Kinase Inhibitors; Rats; Rats, Sprague-Dawley; Sepsis

Severe trauma triggers a systemic inflammatory response that contributes to secondary complications, such as nosocomial infections, sepsis or multi-organ failure. The present study was aimed to identify markers predicting complications and an adverse outcome of severely injured patients by an integrated clinico-transcriptomic approach.. In a prospective study, RNA samples from circulating leukocytes from severely injured patients (injury severity score ≥ 17 points; n = 104) admitted to a Level I Trauma Center were analyzed for dynamic changes in gene expression over a period of 21 days by quantitative RT-PCR. Transcriptomic candidates were selected based on whole genome screening of a representative discovery set (n = 10 patients) or known mechanisms of the immune response, including mediators of inflammation (IL-8, IL-10, TNF-α, MIF, C5, CD59, SPHK1), danger signaling (HMGB1, TLR2, CD14, IL-33, IL-1RL1), and components of the heme degradation pathway (HP, CD163, HMOX1, BLVRA, BLVRB). Clinical markers comprised standard physiological and laboratory parameters and scoring systems routinely determined in trauma patients.. Leukocytes, thrombocytes and the expression of sphingosine kinase-1 (SPHK1), complement C5, and haptoglobin (HP) have been identified as markers with the best performance. Leukocytes showed a biphasic course with peaks on day 0 and day 11 after trauma, and patients with sepsis exhibited significantly higher leukocyte levels. Thrombocyte numbers showed a typical profile with initial thrombopenia and robust thrombocytosis in week 3 after trauma, ranging 2- to 3-fold above the upper normal value. 'Relative thrombocytopenia' was associated with multi-organ dysfunction, the development of sepsis, and mortality, the latter of which could be predicted within 3 days prior to the time point of death. SPHK1 expression at the day of admission indicated mortality with excellent performance. C5-expression on day 1 after trauma correlated with an increased risk for the development of nosocomial infections during the later course, while HP was found to be a marker for the development of sepsis.. The combination of clinical and transcriptomic markers improves the prognostic performance and may represent a useful tool for individual risk stratification in trauma patients.

Topics: Biomarkers; Complement C5; Haptoglobins; Humans; Injury Severity Score; Multiple Organ Failure; Phosphotransferases (Alcohol Group Acceptor); Prospective Studies; Risk Assessment; Sepsis; Systemic Inflammatory Response Syndrome

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

Heat shock proteins (HSPs) are potent protectors of cellular integrity against environmental stresses, including toxic microbial products. To investigate the mechanism of HSP-70 cell protection against bacterial lipopolysaccharide (LPS), we established a stable HSP-70 gene-transfected RAW 264.7 murine macrophage model of LPS-induced cell death. Bacterial LPS increases the activity of sphingosine kinase 1 (SK1), which catalyzes formation of sphingosine-1-phosphate (S1P). S1P functions as a critical signal for initiation and maintenance of diverse aspects of immune cell activation and function. When mouse macrophages were incubated with Escherichia coli LPS (1 microg/ml) and sphingosine kinase inhibitor (SKI, 5 microM), 90% of cells died. Neither LPS nor SKI alone at these doses damaged the cells. The LPS/SKI-induced cell death was partially reversed by overexpression of HSP-70 in gene-transfected macrophages. The specificity of HSP-70 in this reversal was demonstrated by transfection of HSP-70-specific siRNA. Down-regulation of HSP-70 expression after transfection of siRNA specific for HSP-70 was associated with increased LPS/SKI-induced cell damage. Overexpression of human or murine HSP-70 (HSPA1A and Hspa1a, respectively) increased both cellular SK1 mRNA and protein levels. Cellular heat shock also increased SK1 protein. These studies confirm the importance of SK1 as a protective moiety in LPS-induced cell injury and demonstrate that HSP-70-mediated protection from cells treated with LPS/SKI is accompanied by upregulating expression of SK1. HSP-70-mediated increases in SK1 and consequent increased levels of S1P may also play a role in protection of cells from other processes that lead to programmed cell death.

Topics: Animals; Cell Death; Cell Line; Cell Survival; Gene Expression Regulation; Gene Knockdown Techniques; HSP70 Heat-Shock Proteins; Humans; Lipopolysaccharides; Macrophages; Mice; Phosphotransferases (Alcohol Group Acceptor); RNA, Messenger; RNA, Small Interfering; Sepsis; Transfection; Up-Regulation

Activated protein C (APC), the only FDA-approved biotherapeutic drug for sepsis, possesses anticoagulant, antiinflammatory, and barrier-protective activities. However, the mechanisms underlying its anti-inflammatory functions are not well defined. Here, we report that the antiinflammatory activity of APC on macrophages is dependent on integrin CD11b/CD18, but not on endothelial protein C receptor (EPCR). We showed that CD11b/CD18 bound APC within specialized membrane microdomains/lipid rafts and facilitated APC cleavage and activation of protease-activated receptor-1 (PAR1), leading to enhanced production of sphingosine-1-phosphate (S1P) and suppression of the proinflammatory response of activated macrophages. Deletion of the gamma-carboxyglutamic acid domain of APC, a region critical for its anticoagulant activity and EPCR-dependent barrier protection, had no effect on its antiinflammatory function. Genetic inactivation of CD11b, PAR1, or sphingosine kinase-1, but not EPCR, abolished the ability of APC to suppress the macrophage inflammatory response in vitro. Using an LPS-induced mouse model of lethal endotoxemia, we showed that APC administration reduced the mortality of wild-type mice, but not CD11b-deficient mice. These data establish what we believe to be a novel mechanism underlying the antiinflammatory activity of APC in the setting of endotoxemia and provide clear evidence that the antiinflammatory function of APC is distinct from its barrier-protective function and anticoagulant activities.

Topics: 1-Carboxyglutamic Acid; Animals; Endotoxemia; Integrins; Lipopolysaccharides; Lysophospholipids; Membrane Microdomains; Mice; Mice, Inbred C57BL; Mice, Knockout; Phosphotransferases (Alcohol Group Acceptor); Protein C; Sepsis; Sphingosine

During sepsis, activation of phagocytes leads to the overproduction of proinflammatory cytokines, causing systemic inflammation. Despite substantial information regarding the underlying molecular mechanisms that lead to sepsis, several elements in the pathway remain to be elucidated. We found that the enzyme sphingosine kinase 1 (SphK1) is up-regulated in stimulated human phagocytes and in peritoneal phagocytes of patients with severe sepsis. Blockade of SphK1 inhibited phagocyte production of endotoxin-induced proinflammatory cytokines. We observed protection against sepsis in mice treated with a specific SphK1 inhibitor that was enhanced by treatment with a broad-spectrum antibiotic. These results demonstrated a critical role for SphK1 in endotoxin signaling and sepsis-induced inflammatory responses and suggest that inhibition of SphK1 is a potential therapy for septic shock.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Animals; Bacterial Proteins; Cytokines; Endotoxins; Enzyme Activation; Enzyme Inhibitors; Female; Humans; Inflammation; Lipopolysaccharides; Lipoproteins; Macrophages; Macrophages, Peritoneal; Male; Mice; Middle Aged; Neutrophils; NF-kappa B; Peritonitis; Phosphotransferases (Alcohol Group Acceptor); Protein Kinase C-delta; RNA Interference; Sepsis; Shock, Septic; Signal Transduction; Up-Regulation; Young Adult

Each year, more than a half million people in the United States alone die from sepsis, a dire multisystem disease with highly inadequate treatment options. In a recent issue of Science, Puneet and colleagues provide compelling evidence that inhibiting sphingosine kinase 1--an enzyme that resides in immune cells and is activated by inflammatory signals--might have great potential as a therapy for septic shock.

Topics: Enzyme Inhibitors; Humans; Phosphotransferases (Alcohol Group Acceptor); Sepsis; Signal Transduction

Defining critical points of modulation across heterogeneous clinical syndromes may provide insight into new therapeutic approaches. Coagulation initiated by the cytokine-receptor family member known as tissue factor is a hallmark of systemic inflammatory response syndromes in bacterial sepsis and viral haemorrhagic fevers, and anticoagulants can be effective in severe sepsis with disseminated intravascular coagulation. The precise mechanism coupling coagulation and inflammation remains unresolved. Here we show that protease-activated receptor 1 (PAR1) signalling sustains a lethal inflammatory response that can be interrupted by inhibition of either thrombin or PAR1 signalling. The sphingosine 1-phosphate (S1P) axis is a downstream component of PAR1 signalling, and by combining chemical and genetic probes for S1P receptor 3 (S1P3) we show a critical role for dendritic cell PAR1-S1P3 cross-talk in regulating amplification of inflammation in sepsis syndrome. Conversely, dendritic cells sustain escalated systemic coagulation and are the primary hub at which coagulation and inflammation intersect within the lymphatic compartment. Loss of dendritic cell PAR1-S1P3 signalling sequesters dendritic cells and inflammation into draining lymph nodes, and attenuates dissemination of interleukin-1beta to the lungs. Thus, activation of dendritic cells by coagulation in the lymphatics emerges as a previously unknown mechanism that promotes systemic inflammation and lethality in decompensated innate immune responses.

Topics: Animals; Blood Coagulation; Dendritic Cells; Inflammation; Lymphatic System; Mice; Mice, Inbred C57BL; Phosphotransferases (Alcohol Group Acceptor); Receptor Cross-Talk; Receptor, PAR-1; Receptors, Lysosphingolipid; Sepsis; Signal Transduction