Sphingosine 1-Phosphate as Essential Signaling Molecule in Inflammatory Skin Diseases

Sphingolipids are crucial molecules of the mammalian epidermis. The formation of skin-specific ceramides contributes to the formation of lipid lamellae, which are important for the protection of the epidermis from excessive water loss and protect the skin from the invasion of pathogens and the penetration of xenobiotics. In addition to being structural constituents of the epidermal layer, sphingolipids are also key signaling molecules that participate in the regulation of epidermal cells and the immune cells of the skin. While the importance of ceramides with regard to the proliferation and differentiation of skin cells has been known for a long time, it has emerged in recent years that the sphingolipid sphingosine 1-phosphate (S1P) is also involved in processes such as the proliferation and differentiation of keratinocytes. In addition, the immunomodulatory role of this sphingolipid species is becoming increasingly apparent. This is significant as S1P mediates a variety of its actions via G-protein coupled receptors. It is, therefore, not surprising that dysregulation in the signaling pathways of S1P is involved in the pathophysiological conditions of skin diseases. In the present review, the importance of S1P in skin cells, as well as the immune cells of the skin, is elaborated. In particular, the role of the molecule in inflammatory skin diseases will be discussed. This is important because interfering with S1P signaling pathways may represent an innovative option for the treatment of inflammatory skin diseases

Development of Comorbid Depression after Social Fear Conditioning in Mice and Its Effects on Brain Sphingolipid Metabolism

Currently, there are no animal models for studying both specific social fear and social fear with comorbidities. Here, we investigated whether social fear conditioning (SFC), an animal model with face, predictive and construct validity for social anxiety disorder (SAD), leads to the development of comorbidities at a later stage over the course of the disease and how this affects the brain sphingolipid metabolism. SFC altered both the emotional behavior and the brain sphingolipid metabolism in a time-point-dependent manner. While social fear was not accompanied by changes in non-social anxiety-like and depressive-like behavior for at least two to three weeks, a comorbid depressive-like behavior developed five weeks after SFC. These different pathologies were accompanied by different alterations in the brain sphingolipid metabolism. Specific social fear was accompanied by increased activity of ceramidases in the ventral hippocampus and ventral mesencephalon and by small changes in sphingolipid levels in the dorsal hippocampus. Social fear with comorbid depression, however, altered the activity of sphingomyelinases and ceramidases as well as the sphingolipid levels and sphingolipid ratios in most of the investigated brain regions. This suggests that changes in the brain sphingolipid metabolism might be related to the short- and long-term pathophysiology of SAD

Biological Activity of Different Forms of Oxidized Parathyroid Hormone

Preclinical studies have shown that parathyroid hormone (PTH) loses its biological effects through oxidation. PTH can be oxidized at methionines 8 and 18. Three possible variations of oxidized PTH (oxPTH) exist: Met8(ox)PTH, Met18(ox)PTH, and Met8, Met18(di-ox)PTH. A recent study showed that Met18(ox)PTH retained biological activity and was able to upregulate Fgf23 gene expression, whereas Met8(ox)PTH and Met8, Met18(di-ox)PTH showed less or no biological activity. An earlier study likewise showed that the oxidation of Met18 has minor effects on the secondary structure of PTH, whereas the oxidation of Met8 causes substantial structural changes, consistent with another study showing that oxidization just at Met8 blocks the generation of the second messenger cAMP, whereas the effect of the oxidation of Met18 is much less potent in inhibiting cAMP formation. A considerable percentage of circulating PTH in chronic kidney disease (CKD) patients is oxidized. However, we do not know the relative amounts of the different forms of oxPTH with agonistic, partial agonistic, or even antagonistic biological actions in different CKD populations. This might explain different clinical findings in the different CKD populations analyzed so far. The currently available method that was used in these clinical studies just distinguishes between oxPTH and noxPTH without being able to differentiate between different forms of oxPTH. Only methods of PTH measurement that are able to differentiate between PTH forms (noxPTH, Met8(ox)PTH, Met18(ox)PTH, and Met8, Met18(di-ox)PTH) have the potential to improve patient care, because only these methods will definitively separate bioactive from non-bioactive PTH forms. Such methods need to be developed, validated, and used in prospective randomized clinical trials to define the potential value of bioactive PTH forms as a predictor of cardiovascular events, mortality, and bone turnover

Dermal Delivery of the High-Molecular-Weight Drug Tacrolimus by Means of Polyglycerol-Based Nanogels

Polyglycerol-based thermoresponsive nanogels (tNGs) have been shown to have excellent skin hydration properties and to be valuable delivery systems for sustained release of drugs into skin. In this study, we compared the skin penetration of tacrolimus formulated in tNGs with a commercial 0.1% tacrolimus ointment. The penetration of the drug was investigated in ex vivo abdominal and breast skin, while different methods for skin barrier disruption were investigated to improve skin permeability or simulate inflammatory conditions with compromised skin barrier. The amount of penetrated tacrolimus was measured in skin extracts by liquid chromatography tandem-mass spectrometry (LC-MS/MS), whereas the inflammatory markers IL-6 and IL-8 were detected by enzyme-linked immunosorbent assay (ELISA). Higher amounts of tacrolimus penetrated in breast as compared to abdominal skin or in barrier-disrupted as compared to intact skin, confirming that the stratum corneum is the main barrier for tacrolimus skin penetration. The anti-proliferative effect of the penetrated drug was measured in skin tissue/Jurkat cells co-cultures. Interestingly, tNGs exhibited similar anti-proliferative effects as the 0.1% tacrolimus ointment. We conclude that polyglycerol-based nanogels represent an interesting alternative to paraffin-based formulations for the treatment of inflammatory skin conditions

Use of acid ceramidase and sphingosine kinase inhibitors as antiviral compounds against measles virus infection of lymphocytes in vitro

As structural membrane components and signaling effector molecules sphingolipids influence a plethora of host cell functions, and by doing so also the replication of viruses. Investigating the effects of various inhibitors of sphingolipid metabolism in primary human peripheral blood lymphocytes (PBL) and the human B cell line BJAB we found that not only the sphingosine kinase (SphK) inhibitor SKI-II, but also the acid ceramidase inhibitor ceranib-2 efficiently inhibited measles virus (MV) replication. Virus uptake into the target cells was not grossly altered by the two inhibitors, while titers of newly synthesized MV were reduced by approximately 1 log (90%) in PBL and 70–80% in BJAB cells. Lipidomic analyses revealed that in PBL SKI-II led to increased ceramide levels, whereas in BJAB cells ceranib-2 increased ceramides. SKI-II treatment decreased sphingosine-1-phosphate (S1P) levels in PBL and BJAB cells. Furthermore, we found that MV infection of lymphocytes induced a transient (0.5–6 h) increase in S1P, which was prevented by SKI-II. Investigating the effect of the inhibitors on the metabolic (mTORC1) activity we found that ceranib-2 reduced the phosphorylation of p70 S6K in PBL, and that both inhibitors, ceranib-2 and SKI-II, reduced the phosphorylation of p70 S6K in BJAB cells. As mTORC1 activity is required for efficient MV replication, this effect of the inhibitors is one possible antiviral mechanism. In addition, reduced intracellular S1P levels affect a number of signaling pathways and functions including Hsp90 activity, which was reported to be required for MV replication. Accordingly, we found that pharmacological inhibition of Hsp90 with the inhibitor 17-AAG strongly impaired MV replication in primary PBL. Thus, our data suggest that treatment of lymphocytes with both, acid ceramidase and SphK inhibitors, impair MV replication by affecting a number of cellular activities including mTORC1 and Hsp90, which alter the metabolic state of the cells causing a hostile environment for the virus

TSLP is a direct trigger for T cell migration in filaggrin-deficient skin equivalents

Mutations in the gene encoding for filaggrin (FLG) are major predisposing factors for atopic dermatitis (AD). Besides genetic predisposition, immunological dysregulations considerably contribute to its pathophysiology. For example, thymic stromal lymphopoietin (TSLP) is highly expressed in lesional atopic skin and significantly contributes to the pathogenesis of AD by activating dendritic cells that then initiate downstream effects on, for example, T cells. However, little is known about the direct interplay between TSLP, filaggrin-deficient skin and other immune cells such as T lymphocytes. In the present study, FLG knockdown skin equivalents, characterised by intrinsically high TSLP levels, were exposed to activated CD4+ T cells. T cell exposure resulted in an inflammatory phenotype of the skin equivalents. Furthermore, a distinct shift from a Th1/Th17 to a Th2/Th22 profile was observed following exposure of T cells to filaggrin-deficient skin equivalents. Interestingly, TSLP directly stimulated T cell migration exclusively in filaggrin-deficient skin equivalents even in the absence of dendritic cells, indicating a hitherto unknown role of TSLP in the pathogenesis of AD

Being Born Large for Gestational Age is Associated with Increased Global Placental DNA Methylation

Being born small (SGA) or large for gestational age (LGA) is associated with adverse birth outcomes and metabolic diseases in later life of the offspring. It is known that aberrations in growth during gestation are related to altered placental function. Placental function is regulated by epigenetic mechanisms such as DNA methylation. Several studies in recent years have demonstrated associations between altered patterns of DNA methylation and adverse birth outcomes. However, larger studies that reliably investigated global DNA methylation are lacking. The aim of this study was to characterize global placental DNA methylation in relationship to size for gestational age. Global DNA methylation was assessed in 1023 placental samples by LC-MS/MS. LGA offspring displayed significantly higher global placental DNA methylation compared to appropriate for gestational age (AGA; p<0.001). ANCOVA analyses adjusted for known factors impacting on DNA methylation demonstrated an independent association between placental global DNA methylation and LGA births (p<0.001). Tertile stratification according to global placental DNA methylation levels revealed a significantly higher frequency of LGA births in the third tertile. Furthermore, a multiple logistic regression analysis corrected for known factors influencing birth weight highlighted an independent positive association between global placental DNA methylation and the frequency of LGA births (p=0.001)

ST-2191, an Anellated Bismorpholino Derivative of Oxy-Fingolimod, Shows Selective S1P1 Agonist and Functional Antagonist Potency In Vitro and In Vivo.

Sphingosine 1-phosphate (S1P) is an extensively studied signaling molecule that contributes to cell proliferation, survival, migration and other functions through binding to specific S1P receptors. The cycle of S1P1 internalization upon S1P binding and recycling to the cell surface when local S1P concentrations are low drives T cell trafficking. S1P1 modulators, such as fingolimod, disrupt this recycling by inducing persistent S1P1 internalization and receptor degradation, which results in blocked egress of T cells from the secondary lymphoid tissues. The approval of these compounds for the treatment of multiple sclerosis has placed the development of S1PR modulators in the focus of pharmacological research, mostly for autoimmune indications. Here, we report on a novel anellated bismorpholino derivative of oxy-fingolimod, named ST-2191, which exerts selective S1P1 agonist and functional antagonist potency. ST-2191 is also effective in reducing the lymphocyte number in mice, and this effect is not dependent on phosphorylation by sphingosine kinase 2 for activity. These data show that ST-2191 is a novel S1P1 modulator, but further experiments are needed to analyze the therapeutic impact of ST-2191 in animal models of autoimmune diseases

Inverse correlation of intact PTH, oxidized PTH as well as non-oxidized PTH with 25-hydroxyvitamin D3 in kidney transplant recipients

Background 25-hydroxyvitamin D (25(OH)D) and potentially also 1,25-dihydroxyvitamin D (1,25(OH)2D) inhibits the synthesis of parathyroid hormone (PTH) in the chief cells of the parathyroid gland. Clinical studies showing a negative correlation between (25(OH)D and PTH are in good agreement with these findings in basic science studies. However, PTH was measured in these studies with the currently clinically used 2nd or 3rd generation intact PTH (iPTH) assay systems. iPTH assays cannot distinguish between oxidized forms of PTH and non-oxidized PTH. Oxidized forms of PTH are the by far most abundant form of PTH in the circulation of patients with impaired kidney function. Oxidation of PTH causes a loss of function of PTH. Given that the clinical studies done so far were performed with an PTH assay systems that mainly detect oxidized forms of PTH, the real relationship between bioactive non-oxidized PTH and 25(OH)D as well as 1,25(OH)2D is still unknown. Methods To address this topic, we compared for the first time the relationship between 25(OH)D as well as 1,25(OH)2D and iPTH, oxPTH as well as fully bioactive n-oxPTH in 531 stable kidney transplant recipients in the central clinical laboratories of the Charité. Samples were assessed either directly (iPTH) or after oxPTH (n-oxPTH) was removed using a column that used anti-human oxPTH monoclonal antibodies, a monoclonal rat/mouse parathyroid hormone antibody (MAB) was immobilized onto a column with 500 liters of plasma samples. Spearman correlation analysis and Multivariate linear regression were used to evaluate the correlations between the variables. Results There was an inverse correlation between 25(OH)D and all forms of PTH, including oxPTH (iPTH: r=-0.197, p&lt;0.0001; oxPTH: r=-0.203, p&lt;0.0001; n-oxPTH: r=-0.146, p=0.001). No significant correlation was observed between 1,25(OH)2D and all forms of PTH. Multiple linear regression analysis considering age, PTH (iPTH, oxPTH and n-oxPTH), serum calcium, serum phosphor, serum creatinine, fibroblast growth factor 23 (FGF23), osteoprotegerin (OPG), albumin, and sclerostin as confounding factors confirmed these findings. Subgroup analysis showed that our results are not affected by sex and age. Conclusion In our study, all forms of PTH are inversely correlated with 25-hydroxyvitamin D (25(OH)D). This finding would be in line with an inhibition of the synthesis of all forms of PTH (bioactive n-oxPTH and oxidized forms of PTH with minor or no bioactivity) in the chief cells of the parathyroid glad

Sphingosine kinase 1/S1P receptor signaling axis is essential for cellular uptake of Neisseria meningitidis in brain endothelial cells

Invasion of brain endothelial cells (BECs) is central to the pathogenicity of Neisseria meningitidis infection. Here, we established a key role for the bioactive sphingolipid sphingosine-1-phosphate (S1P) and S1P receptor (S1PR) 2 in the uptake process. Quantitative sphingolipidome analyses of BECs infected with N. meningitidis revealed elevated S1P levels, which could be attributed to enhanced expression of the enzyme sphingosine kinase 1 and its activity. Increased activity was dependent on the interaction of meningococcal type IV pilus with the endothelial receptor CD147. Concurrently, infection led to increased expression of the S1PR2. Blocking S1PR2 signaling impaired epidermal growth factor receptor (EGFR) phosphorylation, which has been shown to be involved in cytoskeletal remodeling and bacterial endocytosis. Strikingly, targeting S1PR1 or S1PR3 also interfered with bacterial uptake. Collectively, our data support a critical role of the SphK/S1P/S1PR axis in the invasion of N. meningitidis into BECs, defining a potential target for adjuvant therapy