Intestinal Acid Sphingomyelinase Protects From Severe Pathogen-Driven Colitis

Inflammatory diseases of the gastrointestinal tract are emerging as a global problem with increased evidence and prevalence in numerous countries. A dysregulated sphingolipid metabolism occurs in patients with ulcerative colitis and is discussed to contribute to its pathogenesis. In the present study, we determined the impact of acid sphingomyelinase (Asm), which catalyzes the hydrolysis of sphingomyelin to ceramide, on the course of Citrobacter (C.) rodentium-driven colitis. C. rodentium is an enteric pathogen and induces colonic inflammation very similar to the pathology in patients with ulcerative colitis. We found that mice with Asm deficiency or Asm inhibition were strongly susceptible to C. rodentium infection. These mice showed increased levels of C. rodentium in the feces and were prone to bacterial spreading to the systemic organs. In addition, mice lacking Asm activity showed an uncontrolled inflammatory Th1 and Th17 response, which was accompanied by a stronger colonic pathology compared to infected wild type mice. These findings identified Asm as an essential regulator of mucosal immunity to the enteric pathogen C. rodentium

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

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

Signaling of Sphingosine-1-Phosphate and its influence on endocytotic capacity of dendritic cells.

Sphingosin-1-Phosphat (S1P) stellt einen neuen biologischen Mediator dar, für den eine multilaterale Modulation vieler immunologischer Mechanismen bereits nachgewiesen werden konnte. In diesem Zusammenhang führt die topische Applikation von S1P in einem Tiermodell der experimentellen Atopischen Dermatitis zu einer abgeschwächten Hautentzündungsreaktion. Im Rahmen dieser Arbeit kann zum ersten Mal eine Modulation der dendritischen Zellen (DC`s) durch S1P nachgewiesen werden, die bei einer topischen Behandlung zu einer anti-inflammatorischen Wirkung beiträgt. Diese beruht auf einer Beeinflussung der essentiellen Phase der immunologischen DC-Aktivierung, die durch die Endozytose von Antigenen geprägt ist. Tatsächlich kann eine konzentrationsabhängige Reduktion der endozytotischen Kapazität in unreifen DC`s durch S1P ermittelt werden. In der vorliegenden Arbeit war es zudem von großem Interesse die molekularen Mechanismen aufzuklären, die zu der entsprechenden Reduktion der Endozytose durch S1P führen. Für die Aufnahme von Antigenen durch die unreifen DC`s aus ihrer unmittelbaren Umgebung stehen mehrere Endozytosemechanismen zur Verfügung. Tatsächlich kann für S1P die Reduktion der unspezifischen Flüssigkeitsaufnahme und der darin gelösten Antigene bzw. der Makropinozytose nachgewiesen werden. Die Untersuchung des dafür verantwortlichen Signalwegs zeigt die bereits etablierte Regulation der Phosphoinositid 3-Kinase-Aktivität als Ursache für das veränderte Endozytoseverhalten der unreifen DC`s, welche durch die Stimulation des S1P2-Rezeptorsubtyps bewerkstelligt wird. Darüber hinaus liefert die Erniedrigung der S1P2-Rezeptorexpression einen Hinweis auf eine endogene Regulation der Endozytose durch S1P in unreifen DC`s. So kann im Zuge dieser Arbeit ein autokriner Mechanismus unter Beteiligung des Lipidmediators in den DC`s postuliert werden. Dabei zeigt die Steigerung der endogenen S1P-Synthese mittels pharmakologischer Sphingosinkinase1-Aktivierung die erwartete Reduktion der endozytotischen Kapazität. Die Analyse des für die transmembranäre Translokation von S1P verantwortlichen Mechanismus kann ebenfalls näher identifiziert werden. Der Einsatz verschiedener, spezifischer Hemmstoffe bestätigt die Einbindung des ABCC1-Transporters. Die vorliegenden Untersuchungen zeigen somit eine essentielle Rolle von S1P bei der Endozytose der unreifen DC`s. Da die Antigenaufnahme den Initialschritt in der Immunkaskade darstellt, kann die Aufklärung der damit verbundenen Signalwege zur Etablierung neuer Therapien entzündlicher Erkrankungen wie der Atopischen Dermatitis maßgeblich beitragen.The lipid mediator sphingosine 1-phosphate (S1P) has been identified as a new biological molecule that is involved in the modulation of multilateral immunological processes. In this context, topically administrated S1P inhibits the inflammatory reaction in an animal model of atopic dermatitis. Furthermore, for the first time the present work supplies clear evidence that a modulation of dendritic cell (DC) function contributes to the observed anti- inflammatory effect of local S1P application. The presence of S1P influences the essential step of DC activation, which is characterized by endocytosis of antigens. Stimulation of immature DC`s with S1P demonstrates a dose dependant reduction of antigen capture by these antigen presenting cells. In the present work, it was of great interest to specify the molecular mechanisms that lead to the S1P-induced reduction of endocytosis in immature skin DC`s. In an immature stage, DC`s are able to take up antigens via several different mechanisms. An examination of a mechanism affected by S1P indicated that this sphingolipid inhibits the screening of large volumes of fluid for antigens during macropinocytosis in DC`s. The present study shows that macropinocytosis is mediated by modulation of the PI3K activity, allowing a fine-tuned regulation of antigen capture. Furthermore, the present work indicates that S1P is able to reduce PI3K activity via the S1P2 receptor subtype. Most interestingly, down regulation of S1P2 receptor subtype not only prevents the inhibitory effect of S1P on antigen uptake but also increases the basal level of macropinocytotic capacity. These results provide evidence that S1P could be involved in the endogenous regulation of endocytosis in immature DC`s. This hypothesis has been substantiated by enhancing the endogenous biosynthesis of S1P using a direct inducer of SphK1 activity. As expected, observed reduction of endocytosis by DC`s in the presence of the SphK1 activator was accompanied by diminished phosphoinositid 3-kinase activity. In agreement with previous studies the present work provides evidence, that an ABCC1 transporter is involved in the secretion of the sphingolipid. In summary, the present work demonstrates an essential role of S1P in antigen capture by immature DC`s. Endocytosis is the initial step of the adaptive immune response. Thus, elucidation of related signalling pathways could significantly contribute to the establishment of new treatments for inflammatory diseases such as atopic dermatitis

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

Involvement of Sphingosine 1-Phosphate in Palmitate-Induced Non-Alcoholic Fatty Liver Disease

Background/Aims: Ectopic lipid accumulation in hepatocytes has been identified as a risk factor for the progression of liver fibrosis and is strongly associated with obesity. In particular, the saturated fatty acid palmitate is involved in initiation of liver fibrosis via formation of secondary metabolites by hepatocytes that in turn activate hepatic stellate cells (HSCs) in a paracrine manner. Methods: α-smooth muscle actin-expression (α-SMA) as a marker of liver fibrosis was investigated via western blot analysis and immunofluorescence microscopy in HSCs (LX-2). Sphingolipid metabolism and the generation of the bioactive secondary metabolite sphingosine 1-phosphate (S1P) in response to palmitate were analyzed by LC-MS/MS in hepatocytes (HepG2). To identify the molecular mechanism involved in the progression of liver fibrosis real-time PCR analysis and pharmacological modulation of S1P receptors were performed. Results: Palmitate oversupply increased intra- and extracellular S1P-concentrations in hepatocytes. Conditioned medium from HepG2 cells initiated fibrosis by enhancing α-SMA-expression in LX-2 in a S1P-dependent manner. In accordance, fibrotic response in the presence of S1P was also observed in HSCs. Pharmacological inhibition of S1P receptors demonstrated that S1P3 is the crucial receptor subtype involved in this process. Conclusion: S1P is synthesized in hepatocytes in response to palmitate and released into the extracellular environment leading to an activation of HSCs via the S1P3 receptor

Sphingosine-1-phosphate modulates dendritic cell function : focus on non-migratory effects in vitro and in vivo

Dendritic cells (DCs) are the cutting edge in innate and adaptive immunity. The major functions of these antigen-presenting cells are the capture, endosomal processing and presentation of antigens, providing them an exclusive ability to provoke adaptive immune responses and to induce and control tolerance. Immature DCs capture and process antigens, migrate towards secondary lymphoid organs where they present antigens to naive T cells in a well-synchronized sequence of procedures referred to as maturation. Indeed, recent research indicated that sphingolipids are modulators of essential steps in DC homeostasis. It has been recognized that sphingolipids not only modulate the development of DC subtypes from precursor cells but also influence functional activities of DCs such as antigen capture, and cytokine profiling. Thus, it is not astonishing that sphingolipids and sphingolipid metabolism play a substantial role in inflammatory diseases that are modulated by DCs. Here we highlight the function of sphingosine 1-phosphate (S1P) on DC homeostasis and the role of S1P and S1P metabolism in inflammatory diseases

Inflammatory cells, ceramides, and expression of proteases in perivascular adipose tissue adjacent to human abdominal aortic aneurysms

BACKGROUND: Abdominal aortic aneurysm (AAA) is a deadly irreversible weakening and distension of the abdominal aortic wall. The pathogenesis of AAA remains poorly understood. Investigation into the physical and molecular characteristics of perivascular adipose tissue (PVAT) adjacent to AAA has not been done before and is the purpose of this study. METHODS AND RESULTS: Human aortae, periaortic PVAT, and fat surrounding peripheral arteries were collected from patients undergoing elective surgical repair of AAA. Control aortas were obtained from recently deceased healthy organ donors with no known arterial disease. Aorta and PVAT was found in AAA to larger extent compared with control aortas. Immunohistochemistry revealed neutrophils, macrophages, mast cells, and T-cells surrounding necrotic adipocytes. Gene expression analysis showed that neutrophils, mast cells, and T-cells were found to be increased in PVAT compared with AAA as well as cathepsin K and S. The concentration of ceramides in PVAT was determined using mass spectrometry and correlated with content of T-cells in the PVAT. CONCLUSIONS: Our results suggest a role for abnormal necrotic, inflamed, proteolytic adipose tissue to the adjacent aneurysmal aortic wall in ongoing vascular damage