Lukasz Japtok

Engineered liposomes sequester bacterial exotoxins and protect from severe invasive infections in mice

Gram-positive bacterial pathogens that secrete cytotoxic pore-forming toxins, such as Staphylococcus aureus and Streptococcus pneumoniae, cause a substantial burden of disease. Inspired by the principles that govern natural toxin-host interactions, we have engineered artificial liposomes that are tailored to effectively compete with host cells for toxin binding. Liposome-bound toxins are unable to lyse mammalian cells in vitro. We use these artificial liposomes as decoy targets to sequester bacterial toxins that are produced during active infection in vivo. Administration of artificial liposomes within 10 h after infection rescues mice from septicemia caused by S. aureus and S. pneumoniae, whereas untreated mice die within 24–33 h. Furthermore, liposomes protect mice against invasive pneumococcal pneumonia. Composed exclusively of naturally occurring lipids, tailored liposomes are not bactericidal and could be used therapeutically either alone or in conjunction with antibiotics to combat bacterial infections and to minimize toxin-induced tissue damage that occurs during bacterial clearance.

Hepatocyte exosomes mediate liver repair and regeneration via sphingosine-1-phosphate

BACKGROUND & AIMS Exosomes are small membrane vesicles involved in intercellular communication. Hepatocytes are known to release exosomes, but little is known about their biological function. We sought to determine if exosomes derived from hepatocytes contribute to liver repair and regeneration after injury. METHODS Exosomes derived from primary murine hepatocytes were isolated and characterized biochemically and biophysically. Using cultures of primary hepatocytes, we tested whether hepatocyte exosomes induced proliferation of hepatocytes in vitro. Using models of ischemia/reperfusion injury and partial hepatectomy, we evaluated whether hepatocyte exosomes promote hepatocyte proliferation and liver regeneration in vivo. RESULTS Hepatocyte exosomes, but not exosomes from other liver cell types, induce dose-dependent hepatocyte proliferation in vitro and in vivo. Mechanistically, hepatocyte exosomes directly fuse with target hepatocytes and transfer neutral ceramidase and sphingosine kinase 2 (SK2) causing increased synthesis of sphingosine-1-phosphate (S1P) within target hepatocytes. Ablation of exosomal SK prevents the proliferative effect of exosomes. After ischemia/reperfusion injury, the number of circulating exosomes with proliferative effects increases. CONCLUSIONS Our data shows that hepatocyte-derived exosomes deliver the synthetic machinery to form S1P in target hepatocytes resulting in cell proliferation and liver regeneration after ischemia/reperfusion injury or partial hepatectomy. These findings represent a potentially novel new contributing mechanism of liver regeneration and have important implications for new therapeutic approaches to acute and chronic liver disease.

Sphingoid long chain bases prevent lung infection by Pseudomonas aeruginosa

Cystic fibrosis patients and patients with chronic obstructive pulmonary disease, trauma, burn wound, or patients requiring ventilation are susceptible to severe pulmonary infection by Pseudomonas aeruginosa. Physiological innate defense mechanisms against this pathogen, and their alterations in lung diseases, are for the most part unknown. We now demonstrate a role for the sphingoid long chain base, sphingosine, in determining susceptibility to lung infection by P. aeruginosa. Tracheal and bronchial sphingosine levels were significantly reduced in tissues from cystic fibrosis patients and from cystic fibrosis mouse models due to reduced activity of acid ceramidase, which generates sphingosine from ceramide. Inhalation of mice with sphingosine, with a sphingosine analog, FTY720, or with acid ceramidase rescued susceptible mice from infection. Our data suggest that luminal sphingosine in tracheal and bronchial epithelial cells prevents pulmonary P. aeruginosa infection in normal individuals, paving the way for novel therapeutic paradigms based on inhalation of acid ceramidase or of sphingoid long chain bases in lung infection.

Regulation of hematogenous tumor metastasis by acid sphingomyelinase.

Metastatic dissemination of cancer cells is the ultimate hallmark of malignancy and accounts for approximately 90% of human cancer deaths. We investigated the role of acid sphingomyelinase (Asm) in the hematogenous metastasis of melanoma cells. Intravenous injection of B16F10 melanoma cells into wild‐type mice resulted in multiple lung metastases, while Asm‐deficient mice (Smpd1−/− mice) were protected from pulmonary tumor spread. Transplanting wild‐type platelets into Asm‐deficient mice reinstated tumor metastasis. Likewise, Asm‐deficient mice were protected from hematogenous MT/ret melanoma metastasis to the spleen in a mouse model of spontaneous tumor metastasis. Human and mouse melanoma cells triggered activation and release of platelet secretory Asm, in turn leading to ceramide formation, clustering, and activation of α5β1 integrins on melanoma cells finally leading to adhesion of the tumor cells. Clustering of integrins by applying purified Asm or C16 ceramide to B16F10 melanoma cells before intravenous injection restored trapping of tumor cells in the lung in Asm‐deficient mice. This effect was revertable by arginine‐glycine‐aspartic acid peptides, which are known inhibitors of integrins, and by antibodies neutralizing β1 integrins. These findings indicate that melanoma cells employ platelet‐derived Asm for adhesion and metastasis.

Sphingosine 1-phosphate modulates antigen capture by murine Langerhans cells via the S1P2 receptor subtype.

Dendritic cells (DCs) play a pivotal role in the development of cutaneous contact hypersensitivity (CHS) and atopic dermatitis as they capture and process antigen and present it to T lymphocytes in the lymphoid organs. Recently, it has been indicated that a topical application of the sphingolipid sphingosine 1-phosphate (S1P) prevents the inflammatory response in CHS, but the molecular mechanism is not fully elucidated. Here we indicate that treatment of mice with S1P is connected with an impaired antigen uptake by Langerhans cells (LCs), the initial step of CHS. Most of the known actions of S1P are mediated by a family of five specific G protein-coupled receptors. Our results indicate that S1P inhibits macropinocytosis of the murine LC line XS52 via S1P2 receptor stimulation followed by a reduced phosphatidylinositol 3-kinase (PI3K) activity. As down-regulation of S1P2 not only diminished S1P-mediated action but also enhanced the basal activity of LCs on antigen capture, an autocrine action of S1P has been assumed. Actually, S1P is continuously produced by LCs and secreted via the ATP binding cassette transporter ABCC1 to the extracellular environment. Consequently, inhibition of ABCC1, which decreased extracellular S1P levels, markedly increased the antigen uptake by LCs. Moreover, stimulation of sphingosine kinase activity, the crucial enzyme for S1P formation, is connected not only with enhanced S1P levels but also with diminished antigen capture. These results indicate that S1P is essential in LC homeostasis and influences skin immunity. This is of importance as previous reports suggested an alteration of S1P levels in atopic skin lesions.

Involvement of sphingosine 1-phosphate in palmitate-induced insulin resistance of hepatocytes via the S1P2 receptor subtype

Aims/hypothesisEnhanced plasma levels of NEFA have been shown to induce hepatic insulin resistance, which contributes to the development of type 2 diabetes. Indeed, sphingolipids can be formed via a de novo pathway from the saturated fatty acid palmitate and the amino acid serine. Besides ceramides, sphingosine 1-phosphate (S1P) has been identified as a major bioactive lipid mediator. Therefore, our aim was to investigate the generation and function of S1P in hepatic insulin resistance.MethodsThe incorporation of palmitate into sphingolipids was performed by rapid-resolution liquid chromatography-MS/MS in primary human and rat hepatocytes. The influence of S1P and the involvement of S1P receptors in hepatic insulin resistance was examined in human and rat hepatocytes, as well as in New Zealand obese (NZO) mice.ResultsPalmitate induced an impressive formation of extra- and intracellular S1P in rat and human hepatocytes. An elevation of hepatic S1P levels was observed in NZO mice fed a high-fat diet. Once generated, S1P was able, similarly to palmitate, to counteract insulin signalling. The inhibitory effect of S1P was abolished in the presence of the S1P2 receptor antagonist JTE-013 both in vitro and in vivo. In agreement with this, the immunomodulator FTY720-phosphate, which binds to all S1P receptors except S1P2, was not able to inhibit insulin signalling.Conclusions/interpretationThese data indicate that palmitate is metabolised by hepatocytes to S1P, which acts via stimulation of the S1P2 receptor to impair insulin signalling. In particular, S1P2 inhibition could be considered as a novel therapeutic target for the treatment of insulin resistance.

Divergent role of sphingosine 1-phosphate on insulin resistance.

Insulin resistance is a complex metabolic disorder in which insulin-sensitive tissues fail to respond to the physiological action of insulin. There is a strong correlation of insulin resistance and the development of type 2 diabetes both reaching epidemic proportions. Dysfunctional lipid metabolism is a hallmark of insulin resistance and a risk factor for several cardiovascular and metabolic disorders. Numerous studies in humans and rodents have shown that insulin resistance is associated with elevations of non-esterified fatty acids (NEFA) in the plasma. Moreover, bioactive lipid intermediates such as diacylglycerol (DAG) and ceramides appear to accumulate in response to NEFA, which may interact with insulin signaling. However, recent work has also indicated that sphingosine 1-phosphate (S1P), a breakdown product of ceramide, modulate insulin signaling in different cell types. In this review, we summarize the current state of knowledge about S1P and insulin signaling in insulin sensitive cells. A specific focus is put on the action of S1P on hepatocytes, pancreatic β-cells and skeletal muscle cells. In particular, modulation of S1P-signaling can be considered as a potential therapeutic target for the treatment of insulin resistance and type 2 diabetes.

Sphingosine-1-phosphate exhibits anti-proliferative and anti-inflammatory effects in mouse models of psoriasis.

BACKGROUND It has been indicated that the sphingolipid sphingosine-1-phosphate (S1P) restrains the ability of dendritic cells to migrate to lymph nodes. Furthermore S1P has been demonstrated to inhibit cell growth in human keratinocytes. However, only little is known about the effect of S1P in hyperproliferative and inflammatory in vivo models. OBJECTIVE In this study, locally acting S1P was explored in different experimental mouse models of psoriasis vulgaris. METHODS S1P and FTY720 were tested in the imiquimod-induced psoriasis mouse model, the mouse tail assay and a pilot study of the severe combined immunodeficiency mice (SCID). RESULTS In the imiquimod model the positive control diflorasone diacetate and S1P, but not FTY720 reduced the imiquimod-induced epidermal hyperproliferation of the ear skin. This effect was confirmed in the SCID model, where S1P treated skin from patients suffering from psoriasis showed a decrease in epidermal thickness compared to vehicle. In the imiquimod model, there was also significant inhibition of ear swelling and a moderate reduction of inflammatory cell influx and oedema formation in ear skin by S1P treatment. The inflammatory response on the back skin was, however, only reduced by diflorasone diacetate. In the mouse tail assay, the influence of S1P and FTY720 in stratum granulosum formation was tested compared to the positive control calcipotriol. Whereas topical administration of calcipotriol led to a low but significant increase of stratum granulosum, S1P and FTY720 lacked such an effect. CONCLUSION Taken together, these results imply that topical administration of S1P might be a new option for the treatment of mild to moderate psoriasis lesions.

The ceramide kinase inhibitor NVP-231 inhibits breast and lung cancer cell proliferation by inducing M phase arrest and subsequent cell death

Ceramide kinase (CerK) catalyzes the generation of ceramide‐1‐phosphate which may regulate various cellular functions, including inflammatory reactions and cell growth. Here, we studied the effect of a recently developed CerK inhibitor, NVP‐231, on cancer cell proliferation and viability and investigated the role of cell cycle regulators implicated in these responses.

A sphingolipid mechanism for behavioral extinction

Reward‐dependent instrumental behavior must continuously be re‐adjusted according to environmental conditions. Failure to adapt to changes in reward contingencies may incur psychiatric disorders like anxiety and depression. When an expected reward is omitted, behavior undergoes extinction. While extinction involves active re‐learning, it is also accompanied by emotional behaviors indicative of frustration, anxiety, and despair (extinction‐induced depression). Here, we report evidence for a sphingolipid mechanism in the extinction of behavior. Rapid extinction, indicating efficient re‐learning, coincided with a decrease in the activity of the enzyme acid sphingomyelinase (ASM), which catalyzes turnover of sphingomyelin to ceramide, in the dorsal hippocampus of rats. The stronger the decline in ASM activity, the more rapid was the extinction. Sphingolipid‐focused lipidomic analysis showed that this results in a decline of local ceramide species in the dorsal hippocampus. Ceramides shape the fluidity of lipid rafts in synaptic membranes and by that way can control neural plasticity. We also found that aging modifies activity of enzymes and ceramide levels in selective brain regions. Aging also changed how the chronic treatment with corticosterone (stress) or intranasal dopamine modified regional enzyme activity and ceramide levels, coinciding with rate of extinction. These data provide first evidence for a functional ASM–ceramide pathway in the brain involved in the extinction of learned behavior. This finding extends the known cellular mechanisms underlying behavioral plasticity to a new class of membrane‐located molecules, the sphingolipids, and their regulatory enzymes, and may offer new treatment targets for extinction‐ and learning‐related psychopathological conditions.