Degradation of hexosylceramides is required for timely corpse clearance via formation of cargo-containing phagolysosomal vesicles.

Efficient degradation of phagocytic cargo in lysosomes is crucial to maintain cellular homeostasis and defending cells against pathogens. However, the mechanisms underlying the degradation and recycling of macromolecular cargo within the phagolysosome remain incompletely understood. We previously reported that the phagolysosome containing the corpse of the polar body in C. elegans tubulates into small vesicles to facilitate corpse clearance, a process that requires cargo protein degradation and amino acid export. Here we show that degradation of hexosylceramides by the prosaposin ortholog SPP-10 and glucosylceramidases is required for timely corpse clearance. We observed accumulation of membranous structures inside endolysosomes of spp-10-deficient worms, which are likely caused by increased hexosylceramide species. spp-10 deficiency also caused alteration of additional sphingolipid subclasses, like dihydroceramides, 2-OH-ceramides, and dihydrosphingomyelins. While corpse engulfment, initial breakdown of corpse membrane inside the phagolysosome and lumen acidification proceeded normally in spp-10-deficient worms, formation of the cargo-containing vesicles from the corpse phagolysosome was reduced, resulting in delayed cargo degradation and phagolysosome resolution. Thus, by combining ultrastructural studies and sphingolipidomic analysis with observing single phagolysosomes over time, we identified a role of prosaposin/SPP-10 in maintaining phagolysosomal structure, which promotes efficient resolution of phagocytic cargos.

Caveolin-1 affects early mycobacterial infection and apoptosis in macrophages and mice.

Tuberculosis, caused by Mycobacterium tuberculosis, remains one of the deadliest infections in humans. Because Mycobacterium bovis Bacillus Calmette-Guérin (BCG) share genetic similarities with Mycobacterium tuberculosis, it is often used as a model to elucidate the molecular mechanisms of more severe tuberculosis infection. Caveolin-1 has been implied in many physiological processes and diseases, but it's role in mycobacterial infections has barely been studied. We isolated macrophages from Wildtype or Caveolin-1 deficient mice and analyzed hallmarks of infection, such as internalization, induction of autophagy and apoptosis. For in vivo assays we intravenously injected mice with BCG and investigated tissues for bacterial load with colony-forming unit assays, bioactive lipids with mass spectrometry and changes of protein expressions by Western blotting. Our results revealed that Caveolin-1 was important for early killing of BCG infection in vivo and in vitro, controlled acid sphingomyelinase (Asm)-dependent ceramide formation, apoptosis and inflammatory cytokines upon infection with BCG. In accordance, Caveolin-1 deficient mice and macrophages showed higher bacterial burdens in the livers. The findings indicate that Caveolin-1 plays a role in infection of mice and murine macrophages with BCG, by controlling cellular apoptosis and inflammatory host response. These clues might be useful in the fight against tuberculosis.

The Role of Neutral Sphingomyelinase-2 (NSM2) in the Control of Neutral Lipid Storage in T Cells.

The accumulation of lipid droplets (LDs) and ceramides (Cer) is linked to non-alcoholic fatty liver disease (NAFLD), regularly co-existing with type 2 diabetes and decreased immune function. Chronic inflammation and increased disease severity in viral infections are the hallmarks of the obesity-related immunopathology. The upregulation of neutral sphingomyelinase-2 (NSM2) has shown to be associated with the pathology of obesity in tissues. Nevertheless, the role of sphingolipids and specifically of NSM2 in the regulation of immune cell response to a fatty acid (FA) rich environment is poorly studied. Here, we identified the presence of the LD marker protein perilipin 3 (PLIN3) in the intracellular nano-environment of NSM2 using the ascorbate peroxidase APEX2-catalyzed proximity-dependent biotin labeling method. In line with this, super-resolution structured illumination microscopy (SIM) shows NSM2 and PLIN3 co-localization in LD organelles in the presence of increased extracellular concentrations of oleic acid (OA). Furthermore, the association of enzymatically active NSM2 with isolated LDs correlates with increased Cer levels in these lipid storage organelles. NSM2 enzymatic activity is not required for NSM2 association with LDs, but negatively affects the LD numbers and cellular accumulation of long-chain unsaturated triacylglycerol (TAG) species. Concurrently, NSM2 expression promotes mitochondrial respiration and fatty acid oxidation (FAO) in response to increased OA levels, thereby shifting cells to a high energetic state. Importantly, endogenous NSM2 activity is crucial for primary human CD4+ T cell survival and proliferation in a FA rich environment. To conclude, our study shows a novel NSM2 intracellular localization to LDs and the role of enzymatically active NSM2 in metabolic response to enhanced FA concentrations in T cells.

Evidence of the protective effect of anti-pollution products against oxidative stress in skin ex vivo using EPR spectroscopy and autofluorescence measurements.

The concentration of air pollution is gradually increasing every year so that daily skin exposure is unavoidable. Dietary supplements and topical formulations currently represent the protective strategies to guard against the effects of air pollution on the body and the skin. Unfortunately, there are not yet enough methods available to measure the effectiveness of anti-pollution products on skin. Here, we present two ex vivo methods for measuring the protective effect against air pollution of different cream formulations on the skin: Electron paramagnetic resonance (EPR) spectroscopy and autofluorescence excited by 785 nm using a confocal Raman microspectrometer (CRM). Smoke from one cigarette was used as a model pollutant. EPR spectroscopy enables the direct measurement of free radicals in excised porcine skin after smoke exposure. The autofluorescence in the skin was measured ex vivo, which is an indicator of oxidative stress. Two antioxidants and a chelating agent in a base formulation and a commercial product containing an antioxidant mixture were investigated. The ex vivo studies show that the antioxidant epigallocatechin-3-gallate (EGCG) in the base cream formulation provided the best protection against oxidative stress from smoke exposure for both methods.

Plant defensive responses to insect eggs are inducible by general egg-associated elicitors.

Egg deposition by herbivorous insects is well known to elicit defensive plant responses. Our study aimed to elucidate the insect and plant species specificity of these responses. To study the insect species specificity, we treated Arabidopsis thaliana with egg extracts and egg-associated secretions of a sawfly (Diprion pini), a beetle (Xanthogaleruca luteola) and a butterfly (Pieris brassicae). All egg extracts elicited salicylic acid (SA) accumulation in the plant, and all secretions induced expression of plant genes known to be responsive to the butterfly eggs, among them Pathogenesis-Related (PR) genes. All secretions contained phosphatidylcholine derivatives, known elicitors of SA accumulation and PR gene expression in Arabidopsis. The sawfly egg extract did not induce plant camalexin levels, while the other extracts did. Our studies on the plant species specificity revealed that Solanum dulcamara and Ulmus minor responded with SA accumulation and cell death to P. brassicae eggs, i.e. responses also known for A. thaliana. However, the butterfly eggs induced neoplasms only in S. dulcamara. Our results provide evidence for general, phosphatidylcholine-based, egg-associated elicitors of plant responses and for conserved plant core responses to eggs, but also point to plant and insect species-specific traits in plant-insect egg interactions.

Topical Delivery of Tofacitinib in Dermatology: The Promise of a Novel Therapeutic Class Using Biodegradable Dendritic Polyglycerol Sulfates.

Inflammatory skin diseases, such as psoriasis, atopic dermatitis, and alopecia areata, occur when the regulatory tolerance of the innate immune system is disrupted, resulting in the activation of the Janus kinase-signal transducer and activator of transcription (JAK-STAT) inflammatory signaling pathway by interleukin 6 (IL-6) and other key inflammatory cytokines. JAK inhibitors, such as tofacitinib, bind to these enzymes which are coupled to receptors on cell surfaces and block the transcription of inflammatory cytokine-induced genes. The first topical applications are being marketed, yet insufficient effects regarding indications, such as alopecia areata, suggest that improved delivery technologies could help increase the efficacy. In this study, we used sulfated dendritic polyglycerol with caprolactone segments integrated in its backbone (dPGS-PCL), with a molecular weight of 54 kDa, as a degradable carrier to load and solubilize the hydrophobic drug tofacitinib (TFB). TFB loaded in dPGS-PCL (dPGS-PCL@TFB), at a 11 w/w% loading capacity in aqueous solution, showed in an ex-vivo human skin model better penetration than free TFB in a 30:70 (v/v) ethanol/water mixture. We also investigated the anti-inflammatory efficacy of dPGS-PCL@TFB (0.5 w/w%), dPGS-PCL, and free TFB in the water/ethanol mixture by measuring their effects on IL-6 and IL-8 release, and STAT3 and STAT5 activation in ex vivo skin models of simulated inflamed human skin. Our results suggest that dPGS-PCL@TFB reduces the activation of STAT3 and STAT5 by increasing the penetration of the tofacitinib. However, no statistically significant differences with respect to the inhibition of IL-6 and IL-8 were observed in this short incubation time.

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.

New Therapeutic Options in Pulmonal Diseases: Sphingolipids and Modulation of Sphingolipid Metabolism.

Sphingolipids are crucial molecules in the respiratory airways. As in most other tissues and organs, in the lung sphingolipids play an essential role as structural constituents as they regulate barrier function and fluidity of cell membranes. A lung-specific feature is the occurrence of sphingolipids as minor structural components in the surfactant. However, sphingolipids are also key signaling molecules involved in airway cell signaling and their dynamical formation and metabolism are important for normal lung physiology. Dysregulation of sphingolipid metabolism and signaling is involved in altering lung tissue and initiates inflammatory processes promoting the pathogenesis of pulmonal diseases including cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD), and asthma.In the present review, the important role of specific sphingolipid species in pulmonal diseases will be discussed. Only such an understanding opens up the possibility of developing new therapeutic strategies with the aim of correcting the imbalance in sphingolipid metabolism and signaling. Such delivery strategies have already been studied in animal models of these lung diseases, demonstrating that targeting the sphingolipid profile represents new therapeutic opportunities for lung disorders.

Efficient skin interactions of graphene derivatives: challenge, opportunity or both?

Interactions between graphene, with its wide deployment in consumer products, and skin, the body's largest organ and first barrier, are highly relevant with respect to toxicology and dermal delivery. In this work, interaction of polyglycerol-functionalized graphene sheets, with 200 nm average lateral size and different surface charges, and human skin was studied and their potential as topical delivery systems were investigated. While neutral graphene sheets showed no significant skin interaction, their positively and negatively charged counterparts interacted with the skin, remaining in the stratum corneum. This efficient skin interaction bears a warning but also suggests a new topical drug delivery strategy based on the sheets' high loading capacity and photothermal property. Therefore, the immunosuppressive drug tacrolimus was loaded onto positively and negatively charged graphene sheets, and its release measured with and without laser irradiation using liquid chromatography tandem-mass spectrometry. Laser irradiation accelerated the release of tacrolimus, due to the photothermal property of graphene sheets. In addition, graphene sheets with positive and negative surface charges were loaded with Nile red, and their ability to deliver this cargo through the skin was investigated. Graphene sheets with positive surface charge were more efficient than the negatively charged ones in enhancing Nile red penetration into the skin.

Urinary Excretion of Mercapturic Acids of the Rodent Carcinogen Methyleugenol after a Single Meal of Basil Pesto: A Controlled Exposure Study in Humans.

Methyleugenol (ME), found in numerous plants and spices, is a rodent carcinogen and is classified as "possibly carcinogenic to humans". The hypothesis of a carcinogenic risk for humans is supported by the observation of ME-derived DNA adducts in almost all human liver and lung samples examined. Therefore, a risk assessment of ME is needed. Unfortunately, biomarkers of exposure for epidemiological studies are not yet available. We hereby present the first detection of N-acetyl-l-cysteine conjugates (mercapturic acids) of ME in human urine samples after consumption of a popular ME-containing meal, pasta with basil pesto. We synthesized mercapturic acid conjugates of ME, identified the major product as N-acetyl-S-[3'-(3,4-dimethoxyphenyl)allyl]-l-cysteine (E-3'-MEMA), and developed methods for its extraction and LC-MS/MS quantification in human urine. For conducting an exposure study in humans, a basil cultivar with a suitable ME content was grown for the preparation of basil pesto. A defined meal containing 100 g of basil pesto, corresponding to 1.7 mg ME, was served to 12 participants, who collected the complete urine at defined time intervals for 48 h. Using d6-E-3'-MEMA as an internal standard for LC-MS/MS quantification, we were able to detect E-3'-MEMA in urine samples of all participants collected after the ME-containing meal. Excretion was maximal between 2 and 6 h after the meal and was completed within about 12 h (concentrations below the limit of detection). Excreted amounts were only between 1 and 85 ppm of the ME intake, indicating that the ultimate genotoxicant, 1'-sulfooxy-ME, is formed to a subordinate extent or is not efficiently detoxified by glutathione conjugation and subsequent conversion to mercapturic acids. Both explanations may apply cumulatively, with the ubiquitous detection of ME DNA adducts in human lung and liver specimens arguing against an extremely low formation of 1'-sulfooxy-ME. Taken together, we hereby present the first noninvasive human biomarker reflecting an internal exposure toward reactive ME species.

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.

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<0.0001; oxPTH: r=-0.203, p<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.

Red- and Near-Infrared-Excited Autofluorescence as a Marker for Acute Oxidative Stress in Skin Exposed to Cigarette Smoke Ex Vivo and In Vivo.

Air pollution is increasing worldwide and skin is exposed to high levels of pollution daily, causing oxidative stress and other negative consequences. The methods used to determine oxidative stress in the skin are invasive and non-invasive label-free in vivo methods, which are severely limited. Here, a non-invasive and label-free method to determine the effect of cigarette smoke (CS) exposure on skin ex vivo (porcine) and in vivo (human) was established. The method is based on the measurement of significant CS-exposure-induced enhancement in red- and near-infrared (NIR)-excited autofluorescence (AF) intensities in the skin. To understand the origin of red- and NIR-excited skin AF, the skin was exposed to several doses of CS in a smoking chamber. UVA irradiation was used as a positive control of oxidative stress in the skin. The skin was measured with confocal Raman microspectroscopy before CS exposure, immediately after CS exposure, and after skin cleaning. CS exposure significantly increased the intensity of red- and NIR-excited skin AF in a dose-dependent manner in the epidermis, as confirmed by laser scanning microscopy AF imaging and fluorescence spectroscopy measurements. UVA irradiation enhanced the intensity of AF, but to a lower extent than CS exposure. We concluded that the increase in red- and NIR-excited AF intensities of the skin after CS exposure could clearly be related to the induction of oxidative stress in skin, where skin surface lipids are mainly oxidized.

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.

Parental sex-dependent effects of either maternal or paternal eNOS deficiency on the offspring's phenotype without transmission of the parental eNOS deficiency to the offspring.

Background: Preclinical animal studies and clinical studies indicate that both maternal as well as paternal genetic alterations/gene defects might affect the phenotype of the next-generation without transmissions of the affected gene. Currently, the question of whether the same genetic defect present in the mother or father leads to a similar phenotype in the offspring remains insufficiently elucidated. Methods: In this head-to-head study, we crossbred female and male mice with heterozygous endothelial eNOS knockout (eNOS+/-) with male and female wild-type (wt) mice, respectively. Subsequently, we compared the phenotype of the resulting wt offspring with that of wt offspring born to parents with no eNOS deficiency. Results: Wt female offspring of mothers with heterozygous eNOS showed elevated liver fat accumulation, while wt male offspring of fathers with heterozygous eNOS exhibited increased fasting insulin, heightened insulin levels after a glucose load, and elevated liver glycogen content. By quantitative mass-spectrometry it was shown that concentrations of six serum metabolites (lysoPhosphatidylcholine acyl C20:3, phosphatidylcholine diacyl C36:2, phosphatidylcholine diacyl C38:1, phosphatidylcholine acyl-alkyl C34:1, phosphatidylcholine acyl-alkyl C36:3, and phosphatidylcholine acyl-alkyl C42:5 (PC ae C42:5) as well as four liver carbon metabolites (fructose 6-phosphate, fructose 1,6-bisphosphate, glucose 6-phosphate and fumarate) were different between wt offspring with eNOS+/- mothers and wt offspring with eNOS+/- fathers. Importantly, fumarate was inversely correlated with the liver fat accumulation in female offspring with eNOS+/- mothers and increased liver glycogen in offspring of both sexes with eNOS+/- fathers. The qRT-PCR results revealed that the gene expression patterns were different between wt offspring with eNOS+/- mothers and those offspring with eNOS+/- fathers. Different gene expression patterns were correlated with different observed phenotypic changes in male/female offspring born to mothers or fathers with a heterozygous eNOS genotype. Conclusion: The identical parental genetic alteration (heterozygous eNOS deficiency), without being passed on to the offspring, results in distinct metabolic, liver phenotype, and gene expression pattern variations depending on whether the genetic alteration originated from the father or the mother.

Sphingosine as a New Antifungal Agent against Candida and Aspergillus spp.

This study investigated whether sphingosine is effective as prophylaxis against Aspergillus spp. and Candida spp. In vitro experiments showed that sphingosine is very efficacious against A. fumigatus and Nakeomyces glabrataa (formerly named C. glabrata). A mouse model of invasive aspergillosis showed that sphingosine exerts a prophylactic effect and that sphingosine-treated animals exhibit a strong survival advantage after infection. Furthermore, mechanistic studies showed that treatment with sphingosine leads to the early depolarization of the mitochondrial membrane potential (Δψm) and the generation of mitochondrial reactive oxygen species and to a release of cytochrome C within minutes, thereby presumably initiating apoptosis. Because of its very good tolerability and ease of application, inhaled sphingosine should be further developed as a possible prophylactic agent against pulmonary aspergillosis among severely immunocompromised patients.

The Current Status and Work of Three Rs Centres and Platforms in Europe.

The adoption of Directive 2010/63/EU on the protection of animals used for scientific purposes has given a major push to the formation of Three Rs initiatives in the form of centres and platforms. These centres and platforms are dedicated to the so-called Three Rs, which are the Replacement, Reduction and Refinement of animal use in experiments. ATLA's 50th Anniversary year has seen the publication of two articles on European Three Rs centres and platforms. The first of these was about the progressive rise in their numbers and about their founding history; this second part focuses on their current status and activities. This article takes a closer look at their financial and organisational structures, describes their Three Rs focus and core activities (dissemination, education, implementation, scientific quality/translatability, ethics), and presents their areas of responsibility and projects in detail. This overview of the work and diverse structures of the Three Rs centres and platforms is not only intended to bring them closer to the reader, but also to provide role models and show examples of how such Three Rs centres and platforms could be made sustainable. The Three Rs centres and platforms are very important focal points and play an immense role as facilitators of Directive 2010/63/EU 'on the ground' in their respective countries. They are also invaluable for the wide dissemination of information and for promoting the implementation of the Three Rs in general.

Cell-intrinsic ceramides determine T cell function during melanoma progression.

Acid sphingomyelinase (Asm) and acid ceramidase (Ac) are parts of the sphingolipid metabolism. Asm hydrolyzes sphingomyelin to ceramide, which is further metabolized to sphingosine by Ac. Ceramide generates ceramide-enriched platforms that are involved in receptor clustering within cellular membranes. However, the impact of cell-intrinsic ceramide on T cell function is not well characterized. By using T cell-specific Asm- or Ac-deficient mice, with reduced or elevated ceramide levels in T cells, we identified ceramide to play a crucial role in T cell function in vitro and in vivo. T cell-specific ablation of Asm in Smpd1fl/fl/Cd4cre/+ (Asm/CD4cre) mice resulted in enhanced tumor progression associated with impaired T cell responses, whereas Asah1fl/fl/Cd4cre/+ (Ac/CD4cre) mice showed reduced tumor growth rates and elevated T cell activation compared to the respective controls upon tumor transplantation. Further in vitro analysis revealed that decreased ceramide content supports CD4+ regulatory T cell differentiation and interferes with cytotoxic activity of CD8+ T cells. In contrast, elevated ceramide concentration in CD8+ T cells from Ac/CD4cre mice was associated with enhanced cytotoxic activity. Strikingly, ceramide co-localized with the T cell receptor (TCR) and CD3 in the membrane of stimulated T cells and phosphorylation of TCR signaling molecules was elevated in Ac-deficient T cells. Hence, our results indicate that modulation of ceramide levels, by interfering with the Asm or Ac activity has an effect on T cell differentiation and function and might therefore represent a novel therapeutic strategy for the treatment of T cell-dependent diseases such as tumorigenesis.

Establishment of a method to expose and measure pollution in excised porcine skin with electron paramagnetic resonance spectroscopy.

Health problems associated with the amount of air pollutants are increasing worldwide. Pollution damages not only the lungs; it also has an impact on skin health and is co-responsible for the development of skin diseases. Anti-pollution products are on the rise in the cosmetic market but so far, there is no established method to directly assess the impact of pollution on the skin and to test the efficacy of anti-pollution products. To address this problem, two different chambers were developed for the reproducible exposure to realistic air pollutant concentrations. One chamber for the exclusive use of excised skin and hair samples, the second chamber for ex vivo and in vivo measurements. Measurements of nicotine next to the investigated skin area allow conclusions to be drawn on the particle concentration to which the skin is exposed. Electron paramagnetic resonance spectroscopy, which enables the detection of free radicals in different systems, was applied to assess the hazard potential of pollution in the skin. A direct proof of the formation of free radicals in the skin by the model pollutant cigarette smoke could be demonstrated. An additional application of UV irradiation even increased the formation of free radicals in the skin seven-fold (sum parameter). Depending on the question of interest, the use of different spin probes allows various assessments of the radical formation in skin: the amount of radicals but also the antioxidant status of the microenvironment can be estimated. Using two exposure chambers, the direct formation of oxidative stress by cigarette smoke on ex vivo skin, with and without additional UV exposure, could be reproducibly examined. This measurement method is promising for the assessment of anti-pollution products and could allow a direct causal connection between pollutant, effect on the skin and the protective function of skin care products.

Antiviral and Anti-Inflammatory Activities of Fluoxetine in a SARS-CoV-2 Infection Mouse Model.

The coronavirus disease 2019 (COVID-19) pandemic continues to cause significant morbidity and mortality worldwide. Since a large portion of the world's population is currently unvaccinated or incompletely vaccinated and has limited access to approved treatments against COVID-19, there is an urgent need to continue research on treatment options, especially those at low cost and which are immediately available to patients, particularly in low- and middle-income countries. Prior in vitro and observational studies have shown that fluoxetine, possibly through its inhibitory effect on the acid sphingomyelinase/ceramide system, could be a promising antiviral and anti-inflammatory treatment against COVID-19. In this report, we evaluated the potential antiviral and anti-inflammatory activities of fluoxetine in a K18-hACE2 mouse model of SARS-CoV-2 infection, and against variants of concern in vitro, i.e., SARS-CoV-2 ancestral strain, Alpha B.1.1.7, Gamma P1, Delta B1.617 and Omicron BA.5. Fluoxetine, administrated after SARS-CoV-2 infection, significantly reduced lung tissue viral titres and expression of several inflammatory markers (i.e., IL-6, TNFα, CCL2 and CXCL10). It also inhibited the replication of all variants of concern in vitro. A modulation of the ceramide system in the lung tissues, as reflected by the increase in the ratio HexCer 16:0/Cer 16:0 in fluoxetine-treated mice, may contribute to explain these effects. Our findings demonstrate the antiviral and anti-inflammatory properties of fluoxetine in a K18-hACE2 mouse model of SARS-CoV-2 infection, and its in vitro antiviral activity against variants of concern, establishing fluoxetine as a very promising candidate for the prevention and treatment of SARS-CoV-2 infection and disease pathogenesis.