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Science 328 (5983): 1290-1294

Copyright © 2010 by the American Association for the Advancement of Science

SphK1 Regulates Proinflammatory Responses Associated with Endotoxin and Polymicrobial Sepsis

Padmam Puneet1, Celestial T. Yap1, Lingkai Wong2, Lam Yulin2, Dow Rhoon Koh1, Shabbir Moochhala3, Josef Pfeilschifter4, Andrea Huwiler5, and Alirio J. Melendez1,6,*

1 Department of Physiology, National University of Singapore, 117597 Singapore.
2 Department of Chemistry, National University of Singapore, 117543 Singapore.
3 Defence Medical and Environmental Research Institute, DSO National Laboratories, 117510 Singapore.
4 Pharmazentrum Frankfurt, University Hospital, Frankfurt am Main 60590, Germany.
5 Institute of Pharmacology, University of Bern, Bern CH-3010, Switzerland.
6 Medicine-Immunology, Infection and Inflammation, Glasgow Biomedical Research Centre, University of Glasgow, Glasgow G12 8TA, Scotland, UK.


Figure 1
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Fig. 1. Bacterial products increase human SphK1 expression and function in vitro. (A) Flow cytometric analysis of intracellular SphK1 expression after incubation of human macrophages and neutrophils with LPS and untreated control cells. Isotype control indicates staining of cells with an isotype control antibody. (B) SphK1 expression in human macrophages (top) pretreated with the siRNA-SphK1, scrambled siRNA (siRNA-Scramb.), or with vehicle control (Control PBS). SphK activity in human macrophages stimulated with LPS (bottom) in cells pretreated with vehicle control (PBS), siRNA-SphK1, siRNA-Scramb., or the SphK1-inhibitor 5c. (C) NF-{kappa}B activity in human macrophages stimulated as in (B). (D) Cytokine and HMGB1 production in untreated, nonstimulated human macrophages (Basal) or in human macrophages stimulated as in (B). (E and F) Flow cytometric analysis of SphK1 expression in purified (E) macrophages and (F) neutrophils from patients’ aseptic systemic inflammatory response syndrome (SIRS) from polymicrobial sepsis, and of healthy controls. Isotype control indicates staining of cells with an isotype control antibody. Data shown are representative from 30 septic patients and 15 healthy samples. (G) LPS-mediated cytokines and HMGB1 release by macrophages from septic patients after 16 hours under the indicated stimuation conditions. Basal indicates untreated, unstimulated cells; PBS was used as a vehicle control for 5c; and the cytokines and HMGB1 levels were measured by means of enzyme-linked immunosorbent assay (ELISA). (H) NF-{kappa}B activity in macrophages from septic patients was measured by means of p65 binding to specific DNA templates in an ELISA format. Data are shown as means ± SD (n = 4 separate experiments). Student’s t test P values (*P < 0.05 and **P < 0.01) are compared with LPS-induced control macrophages (LPS+PBS).

 

Figure 2
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Fig. 2. Mechanism for SphK1-mediated, TLR-triggered NF-{kappa}B activation in macrophages from septic patients. (A) Western blot of immunoprecipitates from macrophages stimulated for 10 min under the indicated conditions. Blots were probed for phosphorylated serine/threonine, IKKβ, IKK{alpha}, IKK{gamma}, I{kappa}Bβ, I{kappa}B{alpha}, p65, and PKC{delta}. (B) Total PKC activity was measured by using the Biotrak Protein Kinase C enzyme activity assay (GE Healthcare, UK) in macrophages under the indicated stimulation and pretreatment conditions. (C) NF-{kappa}B activity was measured by means of p65 binding to specific-DNA templates in an ELISA format in human macrophages under the indicated stimulation and pretreatment conditions. (D) Total PKC activity was measured using the Biotrak Protein Kinase C enzyme activity assay in macrophages stimulated with LPS or BLP under the indicated pretreatment conditions. Data are shown as means ± SD from multiple patients (n = 10 patient samples). Student’s t test P values (**P < 0.01) are compared with LPS- or BLP-induced control macrophages (LPS+PBS or BLP+PBS). (E) PKC{delta} is directly activated by the SphK-catalyzed product S1P. Shown are PKC activity assay on recombinant PKC{delta} incubated with PBS (PKC{delta}+PBS), incubated with recombinant SphK1 (PKC{delta}+SphK1), incubated with Sphingosine (PKC{delta}+Sph), incubated with recombinant SphK1 and Sphingosine (PKC{delta}+Sph+SphK1), and incubated with S1P (PKC{delta}+S1P). Data are shown as means ± SD from triplicate measurements from three separate experiments. Student’s t test P values (**P < 0.01) are compared with the control samples (PKCs+PBS).

 

Figure 3
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Fig. 3. Inhibition of SphK1 protects against LPS-induced endotoxic shock in mice. (A) Survival curves for LPS-induced death in mice pretreated with vehicle alone (LPS+PBS), SphK1-siRNA, or scrambled siRNA. As a negative control, mice were pretreated and challenged with vehicle alone (PBS). (B) Serum cytokines or (C) HMGB1 from LPS-injected mice pretreated under the indicated conditions, measured 24 hours after LPS challenge. (D) Immune-cell infiltration and tissue damage in the lungs and liver, 24 hours after LPS challenge, in mice pretreated under the indicated conditions. Immune cells were detected by means of haematoxylin and eosin staining; the magnification is x10. Scale bar, 30 µm. (E) Survival curves for LPS-induced death in the indicated mouse strains in the presence or absence of pretreatment with 2 mg/kg of 5c. (F) LPS-triggered secretion of cytokines and HMGB1 from mouse macrophages from the indicated mouse strains in the presence or absence of pretreatment with 10 µM 5c. Data points correspond to the mean ± SD of three independent experiments (six mice per treatment group; a total of 18 mice per condition were used). Student’s t test P values (**P < 0.01) are compared with LPS-induced control (LPS+PBS).

 

Figure 4
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Fig. 4. Blockade of SphK1 is protective against the CLP model of polymicrobial sepsis. (A to E) Mice were left untreated or pretreated with siRNA-SphK1 (CLP +siRNA-SphK1), scrambled siRNA (CLP +siRNA-Scramb.), 2 mg/kg of 5c (CLP +5c), or vehicle alone (CLP +Vehicle). Sham-operated (Sham) mice were used as a negative control. (A) Survival curves for CLP-induced death. (B) Immune-cell infiltration and tissue damage in the lungs and liver, 24 hours after the CLP, in mice pretreated under the indicated conditions. Immune cells were detected by means of haematoxylin and eosin staining; the magnification was x10. Scale bar, 30 µm. (C) Serum cytokines were measured by means of ELISA 24 hours after CLP. (D) Serum HMGB1 measured 24 hours after CLP. (E) Bacteria in the blood were measured by the colony-forming unit method 24 hours after CLP. Student’s t test P values (**P < 0.01) are compared with CLP-induced control (CLP). (F) Survival in mice treated with 5c (2mg/kg) 2, 6, 8, or 12 hours after CLP. (G) Effects of the antibiotic co-amoxiclav (30 mg/kg) given after 2, 6, 8, or 12 hours on survival after CLP. (H) Survival of mice treated with 5c (2mg/kg) and co-amoxiclav (30 mg/kg) 6, 8, and 12 hours after treatment. Data points correspond to the mean of three independent experiments (n = 6 mice per treatment group).

 


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