A role of sphingolipids for inflammatory bowel disease and cancer is evident. However, the relative and separate contribution of sphingolipid deterioration in inflammation versus carcinogenesis for the pathophysiology of colitis-associated colon cancer (CAC) was unknown and therefore examined in this study. We performed isogenic bone marrow transplantation of inducible sphingosine-1-phosphate (S1P) lyase knockout mice to specifically modulate sphingolipids and associated genes and proteins in a compartment-specific way in a DSS/AOM mediated CAC model. 3D organoid cultures were used in vitro. S1P lyase (SGPL1) knockout in either immune cells or tissue, caused local sphingolipid accumulation leading to a dichotomic development of CAC: Immune cell SGPL1 knockout (I-SGPL −/− ) augmented massive immune cell infiltration initiating colitis with lesions and calprotectin increase. Pathological crypt remodeling plus extracellular S1P-signaling caused delayed tumor formation characterized by S1P receptor 1, STAT3 mRNA increase, as well as programmed cell death ligand 1 expression, accompanied by a putatively counter regulatory STAT1 S727 phosphorylation. In contrast, tissue SGPL1 knockout (T-SGPL −/− ) provoked immediate occurrence of epithelial-driven tumors with upregulated sphingosine kinase 1, S1P receptor 2 and epidermal growth factor receptor. Here, progressing carcinogenesis was accompanied by an IL-12 to IL-23 shift with a consecutive development of a T h 2/GATA3-driven, tumor-favoring microenvironment. Moreover, the knockout models showed distinct lymphopenia and neutrophilia, different from the full SGPL1 knockout. This study shows that depending on the initiating cellular S1P source, the pathophysiology of inflammation-induced cancer versus cancer-induced inflammation develops through separate, discernible molecular steps.

IL-1 family member IL-33 exerts a variety of immune activating and regulating properties and has recently been proposed as a prognostic biomarker for cancer diseases, although its precise role in tumor immunity is unclear. Here we analyzed in vitro conditions influencing the function of IL-33 as an alarmin and a co-factor for the activity of cytotoxic CD8+ T cells in order to explain the widely discussed promiscuous behavior of IL-33 in vivo. Circulating IL-33 detected in the serum of healthy human volunteers was biologically inactive. Additionally, bioactivity of exogenous recombinant IL-33 was significantly reduced in plasma, suggesting local effects of IL-33, and inactivation in blood. Limited availability of nutrients in tissue causes necrosis and thus favors release of IL-33, which-as described before-leads to a locally high expression of the cytokine. The harsh conditions however influence T cell fitness and their responsiveness to stimuli. Nutrient deprivation and pharmacological inhibition of mTOR mediated a distinctive phenotype characterized by expression of IL-33 receptor ST2L on isolated CD8+ T cells, downregulation of CD8, a transitional CD45RAlowROlow phenotype and high expression of secondary lymphoid organ chemokine receptor CCR7. Under nutrient deprivation, IL-33 inhibited an IL-12 induced increase in granzyme B protein expression and increased expression of GATA3 and FOXP3 mRNA. IL-33 enhanced the TCR-dependent activation of CD8+ T cells and co-stimulated the IL-12/TCR-dependent expression of IFNγ. Respectively, GATA3 and FOXP3 mRNA were not regulated during TCR-dependent activation. TCR-dependent stimulation of PBMC, but not LPS, initiated mRNA expression of soluble IL-33 decoy receptor sST2, a control mechanism limiting IL-33 bioactivity to avoid uncontrolled inflammation. Our findings contribute to the understanding of the compartment-specific activity of IL-33. Furthermore, we newly describe conditions, which promote an IL-33-dependent induction of pro- or anti-inflammatory activity in CD8+ T cells during nutrient deprivation.

Systemic sclerosis (SSc) is a rare multi-organ autoimmune disease characterized by progressive skin fibrosis. Inflammation, type 2 immunity, and fibrogenic processes are involved in disease development and may be affected by sphingolipids. However, details about early-stage pathophysiological mechanisms and implicated mediators remain elusive. The sphingolipid sphingosine-1-phosphate (S1P) is elevated in the sera of SSc patients, and its receptor S1P5 is expressed in skin tissue. Nevertheless, almost nothing is known about the dermatological contribution of S1P5 to inflammatory and pro-fibrotic processes leading to the pathological changes seen in SSc. In this study, we observed a novel effect of S1P5 on the inflammatory processes during low-dose bleomycin (BLM)-induced fibrogenesis in murine skin. By comparing 2-week-treated skin areas of wild-type (WT) and S1P5-deficient mice, we found that S1P5 is important for the transcriptional upregulation of the Th2 characteristic transcription factor GATA-3 under treatment-induced inflammatory conditions, while T-bet (Th1) and FoxP3 (Treg) mRNA expression was regulated independently of S1P5. Additionally, treatment caused a regulation of S1P receptor 1 and S1P receptor 3 mRNA as well as a regulation of long-chain ceramide profiles, which both differ significantly between the genotypes. Despite S1P5-dependent differences regarding inflammatory processes, similar macroscopic evidence of fibrosis was detected in the skin histology of WT and S1P5-deficient mice after 4 weeks of subcutaneous BLM treatment. However, at the earlier 2-week point in time, the mRNA data of pro-collagen type 1 and SMAD7 indicate a pro-fibrotic S1P5 contribution in the applied SSc mouse model. In conclusion, we propose that S1P5 plays a role as a novel modulator during the early phase of BLM-caused fibrogenesis in murine skin. An immediate relationship between dermal S1P5 expression and fibrotic processes leading to skin alterations, such as formative for SSc pathogenesis, is indicated but should be studied more profound in further investigations. Therefore, this study is an initial step in understanding the role of S1P5-mediated effects during early stages of fibrogenesis, which may encourage the ongoing search for new therapeutic options for SSc patients.

Objective: A significant proportion of multiple sclerosis (MS) patients respond to treatment with the S1P receptor modulator Fingolimod (FTY720). Following phosphorylation by sphingosinekinase 2 (SphK2) FTY720-P acts as a partial sphingosine-1-phosphate receptor agonist and internalizes sphingosine-1-phosphate receptors, thereby preventing central memory T-cell sequestration. Background: The cytokine IL-33 was recently ascribed a central role in MS as it has been shown to worsen the course of EAE in the mouse model and was detectable in elevated levels peripherally in human plasma and centrally in human normal appearing white matter and MS plaques, whereas IL-33 levels decreased under Interferone-ß-medication. Therefore we aimed at assessing, whether FTY720 might also affect the IL-33 induced T-cell activation. Methods: Primary murine CD8+Tcells were analyzed for FTY720/-P effects on IL-33-induced cytokines. Additionally, serum- and Plasma samples from FTY720 treated MS-patients at steady states were assessed for IL-33 levels and concentrations of 14 bioactive sphingolipids as determined by LC-MS-MS. Fingolimod-treated patients were compared to other treatment groups (n=8-16/group) and healthy controls (HC). Results: IL-33 co-activated CD8+ T-cells, especially by increasing IFN-γ formation in vitro. Interestingly, FTY720, but not FTY720-P, was able to inhibit this IL-33-induced process. Thus, contrainflammatory effects through IL-33-inhibition work independently from intracellular FTY720-phosphorylation by SphK2. Instead, we found FTY720 interacting with the SET/PP2A-pathway. Serum/plasma-samples from MS-patients showed significantly increased S1P- and sphinganin-1-phosphate levels compared to HC. This effect was detectable independently from treatment and Treatment-regime. In a subsequent step, we assess therapy-specific effects on 14 bioactive sphingolipids and IL-33 levels. Conclusions: We show for the first time an alternative, FTY720-phosphorylation-independent immunomodulatory effect of Fingolimod exerted through IL33-induced IFN-γ-Inhibition and signs for comparable human effects. Our data adds to the immunological understanding of therapeutic Fingolimod effects in MS and eventually to decision-making in risk/benefit evaluation when intending to treat a patient with this effective substance.

Multiple sclerosis patients are treated with fingolimod (FTY720), a prodrug that acts as an immune modulator. FTY720 is first phosphorylated to FTY720‐P and then internalizes sphingosine‐1‐phosphate receptors, preventing lymphocyte sequestration. IL‐33 is released from necrotic endothelial cells and contributes to MS severity by coactivating T cells. Herein we analyzed the influence of FTY720, FTY720‐P, and S1P on IL‐33 induced formation of IL‐2 and IFN‐ γ , by using IL‐33 receptor overexpressing EL4 cells, primary CD8 + T cells, and splenocytes. EL4‐ST2 cells released IL‐2 after IL‐33 stimulation that was inhibited dose‐dependently by FTY720‐P but not FTY720. In this system, S1P increased IL‐2, and accordingly, inhibition of S1P producing sphingosine kinases diminished IL‐2 release. In primary CD8 + T cells and splenocytes IL‐33/IL‐12 stimulation induced IFN‐ γ , which was prevented by FTY720 but not FTY720‐P, independently from intracellular phosphorylation. The inhibition of IFN‐ γ by nonphosphorylated FTY720 was mediated via the SET/protein phosphatase 2A (PP2A) pathway, since a SET peptide antagonist also prevented IFN‐ γ formation and the inhibition of IFN‐ γ by FTY720 was reversible by a PP2A inhibitor. While our findings directly improve the understanding of FTY720 therapy in MS, they could also contribute to side effects of FTY720 treatment, like progressive multifocal leukoencephalopathy, caused by an insufficient immune response to a viral infection.

Fingolimod is used for the treatment of multiple sclerosis (MS) and targets receptors for the bioactive sphingolipid sphingosine-1-phosphate (S1P). Whether fingolimod or other MS therapies conversely affect plasma concentrations of sphingolipids has, however, not yet been analyzed. Herein, we quantified 15 representative sphingolipid species by mass spectrometry in plasma from relapsing-remitting MS patients currently under fingolimod (n = 24), natalizumab (n = 16), or IFN-β (n = 18) treatment. Healthy controls (n = 21) and untreated MS patients (n = 11) served as control groups. IFN-ß treatment strongly increased plasma level of C16:0, C18:0, C20:0, and C24:1 ceramides compared to healthy controls, untreated patients, or patients receiving fingolimod or natalizumab medication. Natalizumab treatment increased plasma concentrations of both S1P and sphinganine-1-phosphate, whereas fingolimod treatment did not affect any of these lipids. Correlations of sphingolipids with the Expanded Disability Status Scale and other disease specific parameters revealed no systemic change of sphingolipids in MS, independent of the respective treatment regime. These results indicate type I interferon treatment to cause a strong and specific increase in ceramide level. If confirmed in larger cohorts, these data have implications for the efficacy and adverse effects of IFN-β. Moreover, quantification of ceramides soon after therapy initiation may help to identify therapy-responsive patients.