The sphingosine kinase 1 and S1P1 axis specifically counteracts LPS-induced IL-12p70 production in immune cells of the spleen

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Abstract

Sphingosine-1-phosphate (S1P) has been implicated in angiogenesis, inflammation, cancerogenesis, neurological excitability and immune regulation and is synthesized by two different sphingosine kinases (SphK). It was suggested that mice lacking the gene for SphK1 exhibit no obvious phenotype, because SphK2 compensates for its absence. However, recent investigations revealed that under challenge SphK1 contributed to pro-inflammatory processes favoring Th2 and Th17 rather than Th1-type reactions. To investigate the immune modulatory role of SphK1 as opposed to SphK2 specifically for the Th1 propagating IL-12p70 we compared WT and SphK1−/− splenocytes and Flt3-ligand differentiated BMCs of WT and SphK1−/−, representing dendritic cells as major producers of IL-12p70, incubated with LPS. We determined the impact on IL-12p70 in comparison to other inflammatory cytokines, and on DC and macrophage surface marker expression, SphK mRNA, protein expression and enzymatic activity in splenocytes. Our data demonstrated that SphK1 deficiency enhanced LPS-induced IL-12p70 production although SphK2 was present. To further characterize SphK1-dependent IL-12p70 regulation we exogenously applied S1P, SEW2871 and the new potent S1P1 agonist CYM5442. Both S1P and S1P1-specific analogs fully compensated the increase of IL-12p70 production in SphK1-deficient splenocytes. The use of pertussis toxin, to block Gi-coupled signaling downstream of S1P1, again increased IL-12p70 and neglected the compensation achieved by addition of S1P and S1P1 agonists pointing on the importance of this specific S1P-receptor. Given that, in parallel to a prominent IL-12p35 increase following LPS stimulation, LPS also enhanced SphK expression and total SphK activity, we concluded that SphK1-derived S1P acting via S1P1 is a major mechanism of this negative IL-12p70 feedback loop, which did not affect other cytokines. Moreover, our data showed that SphK2 activity failed to compensate for SphK1 deficiency. These findings clearly point to a divergent and cytokine-specific impact of immune cell SphK1 and SphK2 in chronic inflammation and cancer.

Introduction

Sphingosine kinase (Sphk) 1 is a highly conserved enzyme that catalyzes the conversion of sphingosine to sphingosine-1-phosphate (S1P) (Vadas et al., 2008). The mSphK1 gene is located on chromosome 11 E2 and encodes for a 43-kDa protein for which the highest expression levels are found in the lungs, liver, and spleen (Wadgaonkar et al., 2009). Deletion mutant mice for SphK1 showed no obvious phenotype. These findings led to the assumption that the second SphK subtype, SphK2, which is located on chromosome 7 and exhibits different enzymatic kinetic properties (Liu et al., 2000) and substrate specificity (Olivera et al., 1998, Wattenberg et al., 2006), may functionally compensate for the lack of SphK1; this however has only been shown during embryogenesis and under resting conditions. SphK1 and SphK2 are also located in different compartments inside the cell. Several studies have shown that SphK1 is activated rapidly by cytokines and growth factors (Cuvillier et al., 1996) and then translocates to the plasma membrane or to the outer nucleus membrane which has not been observed for SphK2 (Alemany et al., 2007, Kleuser et al., 2001). Recent studies suggested an involvement of calcium signaling (Ghosh et al., 1990), MAPK (Bu et al., 2006) and the LPS-TLR pathway for the induction of SphK1 expression (Wadgaonkar et al., 2009). Together with other studies showing a coordinated regulation of SphK1 in parallel to external and internal stimuli, this points to a modulatory role either directly of SphK1 or via its product S1P.

Since the first description of its effect in cellular signaling in 1991 (Zhang et al., 1991) the knowledge about S1P as extra- and intracellular messenger has grown exponentially. S1P, generated by either of the two kinases, has pleiotropic effects within several different tissues. The extra- and intracellular sphingolipid levels are additionally regulated by catabolic enzymes dephosporylating or cleaving S1P (Brindley, 2004, Melendez, 2008, Saba and Hla, 2004). Its prominent role for immune regulation has been uncovered by its partial analog FTY720 (Kappos et al., 2010) and more recent studies with S1P-lyase−/−, S1P-lyase−/+ and humanized S1P-lyaseh/h mice showing different lyase activities (Vogel et al., 2009). While rescue of S1P-lyase deficiency with hS1P-lyase could compensate the lethal non-lymphoid lesion formation, T cell development and trafficking could not be restored, indicating that immunologic processes may be more sensitive than others. S1P is predominantly produced and secreted by macrophages, platelets, mast cells and dendritic cells (DC) (Goetzl et al., 2004). Whereas increasing evidence of intracellular functions/targets for S1P exist (Alvarez et al., 2010, Hait et al., 2009, Itagaki et al., 2007, Olivera and Spiegel, 1993) the most well-characterized functions of S1P are mediated via five different G-protein-coupled-receptors (GPCR) named S1P1-5 (Spiegel and and Milstien, 2003). Thus depending on the subset of receptors, which are expressed on the surface of the cell, different intracellular pathways are triggered upon S1P-binding, regulating physiological and pathophysiological functions.

Research on the function of SphK/S1P in different immune cells or its involvement in clinical relevant diseases like colitis, rheumatoid arthritis and sepsis have shown that SphK activity, and subsequently altered S1P concentrations, can not easily be attributed to either pro- or anti-inflammatory responses. While SphK1, rather than SphK2 activity, has been linked to the release of pro-inflammatory cytokines such as IL-1β, IL-6 and TNFα in monocytes (Puneet et al., 2010, Zhi et al., 2006) and macrophages (Puneet et al., 2010, Yadav et al., 2006) and also promoted inflammation in rheumatoid arthritis (Baker et al., 2010, Kitano et al., 2006), colitis (Maines et al., 2008) and sepsis models (Puneet et al., 2010) contrasting investigations exist showing e.g. that Th1 cells overexpressing SphK1 produced less IL-2, IFNγ and TNFα (Wang et al., 2005, Yang et al., 2005).

The spleen is the largest secondary lymphoid organ containing about one-fourth of the body's lymphocytes and is involved in immune responses to blood-derived microbes (Nolte et al., 2002). Thus, given the conflicting results regarding a pro- or anti-inflammatory function of SphK1 and its product S1P we sought to analyze in detail their role for the specific DC cytokine IL-12p70 compared to other above mentioned cytokines in this organ. Therefore, we used SphK1-deficient mice and S1P1-specific agonists to investigate in detail how SphK1 and the product derived from its activity, S1P, and the subsequent S1P signaling affects TLR ligand-induced IL-12p35 and IL-12p40 expression of DCs in a complex mixture of splenocytes and in Flt3-ligand differentiated CD8α+ cDCs which are supposed to be the main producers of biological active IL-12p70 (Shortman and Heath, 2010). Herein, we will demonstrate that SphK1 deficiency enhanced LPS-induced IL-12p70 but not IL-6 or IL-10 production, and that S1P1-specific agonists are sufficient to compensate for SphK1 deficiency in spleen-derived immune cells.

Section snippets

Preparation and cultivation of splenocytes and bone marrow cells

WT C57BL/6 mice were purchased from Harlan Winkelmann; SphK1 knockout (SphK1−/−) mice, backcrossed to the C57BL/6 background were bred at the local animal facility under specific pathogen-free conditions.

Whole spleens were dissected from mice, gently homogenized with a glass tissue homogenizer, and cultured in Iscove‘s medium supplemented with 5% FCS (PAA Laboratories), 2 mM l-glutamine, 100 IU/ml penicillin, 100 μg/ml streptomycin, 1 mM sodium pyruvate, 100 μM nonessential amino acids and 50 μM 2-ME

Genetic deletion of SphK1 and its impact on TLR-dependent activation of immune cells

In recent years several studies have reported about the important role of S1P in various clinical aspects of diseases especially by preventing apoptosis and inducing cell growth and consequently promoting cancer growth and progression (see Section 1). Additionally, it has been shown that SphK1 but not SphK2 is upregulated in many cancer tissues. Given these divergent characteristics of SphK1 and SphK2 and the suggested opposing function of IL-12p70 and IL-23 in inflammation-derived cancer, we

Discussion

The immune suppressive effects of disrupting the gradient of S1P concentrations have been well established, e.g. in governing the lymphocyte migration between blood and secondary lymphatic organs or within the thymus with subsequent lymphocyte sequestration (Mandala et al., 2002). All three of the major S1P level determining sphingolipid enzymes, i.e. S1P lyase, sphingosine kinase-1 and -2 are involved. However recently additional and more direct cytokine or gene regulatory functions of both

Conflict of interest

The authors have no financial conflict of interest.

Acknowledgements

We like to thank Dagmar Meyer zu Heringdorf for their valuable and thoughtful input, as well as Alexander Koch for his support in animal supply.

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