Identification of Bcl-6-dependent follicular helper NKT cells that provide cognate help for B cell responses.

Lipid antigens trigger help from natural killer T cells (NKT cells) for B cells, and direct conjugation of lipid agonists to antigen profoundly augments antibody responses. Here we show that in vivo, NKT cells engaged in stable and prolonged cognate interactions with B cells and induced the formation of early germinal centers. Mouse and human NKT cells formed CXCR5(+)PD-1(hi) follicular helper NKT cells (NKT(FH) cells), and this process required expression of the transcriptional repressor Bcl-6, signaling via the coreceptor CD28 and interaction with B cells. NKT(FH) cells provided direct cognate help to antigen-specific B cells that was dependent on interleukin 21 (IL-21). Unlike T cell-dependent germinal centers, those driven by NKT(FH) cells did not generate long-lived plasma cells. Our results demonstrate the existence of a Bcl-6-dependent subset of NKT cells specialized in providing help to B cells.

A single subset of dendritic cells controls the cytokine bias of natural killer T cell responses to diverse glycolipid antigens.

Many hematopoietic cell types express CD1d and are capable of presenting glycolipid antigens to invariant natural killer T cells (iNKT cells). However, the question of which cells are the principal presenters of glycolipid antigens in vivo remains controversial, and it has been suggested that this might vary depending on the structure of a particular glycolipid antigen. Here we have shown that a single type of cell, the CD8α(+) DEC-205(+) dendritic cell, was mainly responsible for capturing and presenting a variety of different glycolipid antigens, including multiple forms of α-galactosylceramide that stimulate widely divergent cytokine responses. After glycolipid presentation, these dendritic cells rapidly altered their expression of various costimulatory and coinhibitory molecules in a manner that was dependent on the structure of the antigen. These findings show flexibility in the outcome of two-way communication between CD8α(+) dendritic cells and iNKT cells, providing a mechanism for biasing toward either proinflammatory or anti-inflammatory responses.

Pharmacological sphingosine-1 phosphate receptor 1 targeting in cigarette smoke-induced emphysema in mice.

Primarily caused by chronic cigarette smoking (CS), emphysema is characterized by loss of alveolar cells comprising lung units involved in gas exchange and inflammation that culminate in airspace enlargement. Dysregulation of sphingolipid metabolism with increases of ceramide relative to sphingosine-1 phosphate (S1P) signaling has been shown to cause lung cell apoptosis and is emerging as a potential therapeutic target in emphysema. We sought to determine the impact of augmenting S1P signaling via S1P receptor 1 (S1P1) in a mouse model of CS-induced emphysema. DBA2 mice were exposed to CS for 4 or 6 mo and treated with pharmacological agonists of S1P1: phosphonated FTY720 (FTY720-1S and 2S analogs; 0.01-1.0 mg/kg) or GSK183303A (10 mg/kg). Pharmacological S1P1 agonists ameliorated CS-induced lung parenchymal apoptosis and airspace enlargement as well as loss of body weight. S1P1 agonists had modest inhibitory effects on CS-induced airspace inflammation and lung functional changes measured by Flexivent, improving lung tissue resistance. S1P1 abundance was reduced in chronic CS-conditions and remained decreased after CS-cessation or treatment with FTY720-1S. These results support an important role for S1P-S1P1 axis in maintaining the structural integrity of alveoli during chronic CS exposure and suggest that increasing both S1P1 signaling and abundance may be beneficial to counteract the effects of chronic CS exposure.

The assembly of glycosphingolipid determines their immunomodulatory effect: A novel method for structure-based design of immunotherapy.

Structure-activity relationships provide insight into the binding interactions of beta-glycosphingolipids (GSLs) with both the TCR and the CD1d molecules, as well as the subsequent immunologic response of regulatory NKT cells. AIM: To determine the effects of synthetic GSL structures on their immune modulatory functions. METHODS: GSLs of various structures were tested in vitro and in an animal model of Concanavalin A (ConA) immune-mediated hepatitis. RESULTS: In vitro, using SV40 binding to live monkey CV1 cells, the l-threo stereoisomer of C8-ß-LacCer inhibits caveolar internalization, reducing viral binding to the cell surface. In vivo, in the ConA model, LR172, which has a saturated C8 chain, and LR178, which has a trans double bond at C-2 in the C8 chain, suppressed the immune-mediated liver inflammation and reduced IFNγ levels in a dose dependent manner. The beneficial effects of LR172 and of LR178 are associated with suppression of liver apoptosis, increased phosphorylated STAT3 expression in the liver, and an increase in the NKT liver/spleen ratio. SUMMARY: The assembly of GSLs determines their immunomodulatory effect and can serve as a method for structure-based design of immunotherapy.

DHA Modifies the Size and Composition of Raftlike Domains: A Solid-State 2H NMR Study.

Docosahexaenoic acid is an omega-3 polyunsaturated fatty acid that relieves the symptoms of a wide variety of chronic inflammatory disorders. The structural mechanism is not yet completely understood. Our focus here is on the plasma membrane as a site of action. We examined the molecular organization of [2H31]-N-palmitoylsphingomyelin (PSM-d31) mixed with 1-palmitoyl-2-docosahexaenoylphosphatylcholine (PDPC) or 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), as a monounsaturated control, and cholesterol (chol) (1:1:1 mol) in a model membrane by solid-state 2H NMR. The spectra were analyzed in terms of segregation into ordered SM-rich/chol-rich (raftlike) and disordered PC-rich/chol-poor (nonraft) domains that are nanoscale in size. An increase in the size of domains is revealed when POPC was replaced by PDPC. Spectra that are single-component, attributed to fast exchange between domains (<45 nm), for PSM-d31 mixed with POPC and chol become two-component, attributed to slow exchange between domains (r > 30 nm), for PSM-d31 mixed with PDPC and chol. The resolution of separate signals from PSM-d31, and correspondingly from [3α-2H1]cholesterol (chol-d1) and 1-[2H31]palmitoyl-2-docosahexaenoylphosphatidylcholine (PDPC-d31), in raftlike and nonraft domains enabled us to determine the composition of the domains in the PDPC-containing membrane. Most of the lipid (28% SM, 29% chol, and 23% PDPC with respect to total lipid at 30°C) was found in the raftlike domain. Despite substantial infiltration of PDPC into raftlike domains, there appears to be minimal effect on the order of SM, implying the existence of internal structure that limits contact between SM and PDPC. Our results suggest a significant refinement to the model by which DHA regulates the architecture of ordered, sphingolipid-chol-enriched domains (rafts) in membranes.

Sphingosine 1-Phosphate Activation of EGFR As a Novel Target for Meningitic Escherichia coli Penetration of the Blood-Brain Barrier.

Central nervous system (CNS) infection continues to be an important cause of mortality and morbidity, necessitating new approaches for investigating its pathogenesis, prevention and therapy. Escherichia coli is the most common Gram-negative bacillary organism causing meningitis, which develops following penetration of the blood-brain barrier (BBB). By chemical library screening, we identified epidermal growth factor receptor (EGFR) as a contributor to E. coli invasion of the BBB in vitro. Here, we obtained the direct evidence that CNS-infecting E. coli exploited sphingosine 1-phosphate (S1P) for EGFR activation in penetration of the BBB in vitro and in vivo. We found that S1P was upstream of EGFR and participated in EGFR activation through S1P receptor as well as through S1P-mediated up-regulation of EGFR-related ligand HB-EGF, and blockade of S1P function through targeting sphingosine kinase and S1P receptor inhibited EGFR activation, and also E. coli invasion of the BBB. We further found that both S1P and EGFR activations occurred in response to the same E. coli proteins (OmpA, FimH, NlpI), and that S1P and EGFR promoted E. coli invasion of the BBB by activating the downstream c-Src. These findings indicate that S1P and EGFR represent the novel host targets for meningitic E. coli penetration of the BBB, and counteracting such targets provide a novel approach for controlling E. coli meningitis in the era of increasing resistance to conventional antibiotics.

LAPTM4B facilitates late endosomal ceramide export to control cell death pathways.

Lysosome-associated protein transmembrane-4b (LAPTM4B) associates with poor prognosis in several cancers, but its physiological function is not well understood. Here we use novel ceramide probes to provide evidence that LAPTM4B interacts with ceramide and facilitates its removal from late endosomal organelles (LEs). This lowers LE ceramide in parallel with and independent of acid ceramidase-dependent catabolism. In LAPTM4B-silenced cells, LE sphingolipid accumulation is accompanied by lysosomal membrane destabilization. However, these cells resist ceramide-driven caspase-3 activation and apoptosis induced by chemotherapeutic agents or gene silencing. Conversely, LAPTM4B overexpression reduces LE ceramide and stabilizes lysosomes but sensitizes to drug-induced caspase-3 activation. Together, these data uncover a cellular ceramide export route from LEs and identify LAPTM4B as its regulator. By compartmentalizing ceramide, LAPTM4B controls key sphingolipid-mediated cell death mechanisms and emerges as a candidate for sphingolipid-targeting cancer therapies.

Structure and dynamics of the aliphatic cholesterol side chain in membranes as studied by (2)H NMR spectroscopy and molecular dynamics simulation.

Cholesterol is an evolutionarily highly optimized molecule particularly known for its ability to condense the phospholipids in cellular membranes. Until recently, the accompanying increase in the chain order of the surrounding phospholipids was attributed to the planar and rigid tetracyclic ring structure of cholesterol. However, detailed investigations of cholesterol's aliphatic side chain demonstrated that this side chain is responsible for approximately half of the condensation effect. Therefore, we investigated the structure and dynamics of the aliphatic side chain of cholesterol using (2)H solid-state nuclear magnetic resonance (NMR) spectroscopy and microsecond timescale all-atom molecular dynamics (MD) simulations in four different model membranes: POPC, DPPC, PSM, and POPC/PSM (1 : 1 mol/mol) and at three different temperatures: 5 °C, 37 °C, and 50 °C. A cholesterol variant, in which 11 hydrogens of the aliphatic side chain were exchanged for deuterium, was used and the respective (2)H NMR spectra confirmed the axially asymmetric rotational diffusion of cholesterol in DPPC and PSM. Furthermore, NMR spectra indicated that some hydrogens showed an unexpected magnetic inequivalency. This finding was confirmed by all-atom molecular dynamics simulations and detailed analysis revealed that the hydrogens of the methylene groups at C22, C23, and C24 are magnetically inequivalent. This inequivalency is caused by steric clashes of the aliphatic side chain with the ring structure of cholesterol as well as the branched C21 methyl group. These excluded volume effects result in reduced conformational flexibility of the aliphatic side chain of cholesterol and explain its high order (order parameter of 0.78 for chain motions) and large contribution to the condensation effect. Additionally, the motional pattern of the side chain becomes highly anisotropic such that it shows larger fluctuations perpendicular to the ring plane of cholesterol with a biaxiality of the distribution of 0.046. Overall, our results shed light on the mechanism how the aliphatic side chain is able to contribute about half of the condensation effect of cholesterol.

Inhibition of mTOR down-regulates scavenger receptor, class B, type I (SR-BI) expression, reduces endothelial cell migration and impairs nitric oxide production.

The mammalian target of rapamycin (mTOR) inhibiting drug rapamycin (Sirolimus) has severe side effects in patients including hyperlipidemia, an established risk factor for atherosclerosis. Recently, it was shown that rapamycin decreases hepatic LDL receptor (LDL-R) expression, which likely contributes to hypercholesterolemia. Scavenger receptor, class B, type I (SR-BI) is the major HDL receptor and consequently regulating HDL-cholesterol levels and the athero-protective effects of HDL. By using the mTOR inhibitor rapamycin, we show that SR-BI is down-regulated in human umbilical vein endothelial cells (HUVECs). This reduction of SR-BI protein as well as mRNA levels by about 50% did not alter HDL particle uptake or HDL-derived lipid transfer. However, rapamycin reduced HDL-induced activation of eNOS and stimulation of endothelial cell migration. The effects on cell migration could be counteracted by SR-BI overexpression, indicating that decreased SR-BI expression is in part responsible for the rapamycin-induced effects. We demonstrate that inhibition of mTOR leads to endothelial cell dysfunction and decreased SR-BI expression, which may contribute to atherogenesis during rapamycin treatment.

Endothelial disruptive proinflammatory effects of nicotine and e-cigarette vapor exposures.

The increased use of inhaled nicotine via e-cigarettes has unknown risks to lung health. Having previously shown that cigarette smoke (CS) extract disrupts the lung microvasculature barrier function by endothelial cell activation and cytoskeletal rearrangement, we investigated the contribution of nicotine in CS or e-cigarettes (e-Cig) to lung endothelial injury. Primary lung microvascular endothelial cells were exposed to nicotine, e-Cig solution, or condensed e-Cig vapor (1-20 mM nicotine) or to nicotine-free CS extract or e-Cig solutions. Compared with nicotine-containing extract, nicotine free-CS extract (10-20%) caused significantly less endothelial permeability as measured with electric cell-substrate impedance sensing. Nicotine exposures triggered dose-dependent loss of endothelial barrier in cultured cell monolayers and rapidly increased lung inflammation and oxidative stress in mice. The endothelial barrier disruptive effects were associated with increased intracellular ceramides, p38 MAPK activation, and myosin light chain (MLC) phosphorylation, and was critically mediated by Rho-activated kinase via inhibition of MLC-phosphatase unit MYPT1. Although nicotine at sufficient concentrations to cause endothelial barrier loss did not trigger cell necrosis, it markedly inhibited cell proliferation. Augmentation of sphingosine-1-phosphate (S1P) signaling via S1P1 improved both endothelial cell proliferation and barrier function during nicotine exposures. Nicotine-independent effects of e-Cig solutions were noted, which may be attributable to acrolein, detected along with propylene glycol, glycerol, and nicotine by NMR, mass spectrometry, and gas chromatography, in both e-Cig solutions and vapor. These results suggest that soluble components of e-Cig, including nicotine, cause dose-dependent loss of lung endothelial barrier function, which is associated with oxidative stress and brisk inflammation.

NDRG1 functions in LDL receptor trafficking by regulating endosomal recycling and degradation.

N-myc downstream-regulated gene 1 (NDRG1) mutations cause Charcot-Marie-Tooth disease type 4D (CMT4D). However, the cellular function of NDRG1 and how it causes CMT4D are poorly understood. We report that NDRG1 silencing in epithelial cells results in decreased uptake of low-density lipoprotein (LDL) due to reduced LDL receptor (LDLR) abundance at the plasma membrane. This is accompanied by the accumulation of LDLR in enlarged EEA1-positive endosomes that contain numerous intraluminal vesicles and sequester ceramide. Concomitantly, LDLR ubiquitylation is increased but its degradation is reduced and ESCRT (endosomal sorting complex required for transport) proteins are downregulated. Co-depletion of IDOL (inducible degrader of the LDLR), which ubiquitylates the LDLR and promotes its degradation, rescues plasma membrane LDLR levels and LDL uptake. In murine oligodendrocytes, Ndrg1 silencing not only results in reduced LDL uptake but also in downregulation of the oligodendrocyte differentiation factor Olig2. Both phenotypes are rescued by co-silencing of Idol, suggesting that ligand uptake through LDLR family members controls oligodendrocyte differentiation. These findings identify NDRG1 as a novel regulator of multivesicular body formation and endosomal LDLR trafficking. The deficiency of functional NDRG1 in CMT4D might impair lipid processing and differentiation of myelinating cells.

Ceramide-1-phosphate inhibits cigarette smoke-induced airway inflammation.

Sphingolipids are involved in the pathogenesis of inflammatory diseases. The central molecule is ceramide, which can be converted into ceramide-1-phosphate (C1P). Although C1P can exert anti- and pro-inflammatory effects, its influence on cigarette smoke (CS)-induced lung inflammation is unknown. We aimed to clarify the role of C1P in the pathogenesis of CS-triggered pulmonary inflammation and emphysema in humans and mice. The effects of C1P were addressed on CS-induced lung inflammation in C57BL/6 mice, CS extract-triggered activation of human airway epithelial cells (AECs) and neutrophils from patients with chronic obstructive pulmonary disease. Differential cell counts in bronchoalveolar lavage fluid were determined by flow cytometry and pro-inflammatory cytokines were measured by ELISA. Expression and DNA binding of nuclear factor (NF)-κB and neutral sphingomyelinase (nSMase) were quantified by PCR, electrophoretic mobility shift and fluorometric assays. C1P reduced CS-induced acute and chronic lung inflammation and development of emphysema in mice, which was associated with a reduction in nSMase and NF-κB activity in the lungs. nSMase activity in human serum correlated negatively with forced expiratory volume in 1 s % predicted. In human AECs and neutrophils, C1P inhibited CS-induced activation of NF-κB and nSMase, and reduced pro-inflammatory cytokine release. Our results suggest that C1P is a potential target for anti-inflammatory treatment in CS-induced lung inflammation.

Antagonistic activities of the immunomodulator and PP2A-activating drug FTY720 (Fingolimod, Gilenya) in Jak2-driven hematologic malignancies.

FTY720 (Fingolimod, Gilenya) is a sphingosine analog used as an immunosuppressant in multiple sclerosis patients. FTY720 is also a potent protein phosphatase 2A (PP2A)-activating drug (PAD). PP2A is a tumor suppressor found inactivated in different types of cancer. We show here that PP2A is inactive in polycythemia vera (PV) and other myeloproliferative neoplasms characterized by the expression of the transforming Jak2(V617F) oncogene. PP2A inactivation occurs in a Jak2(V617F) dose/kinase-dependent manner through the PI-3Kγ-PKC-induced phosphorylation of the PP2A inhibitor SET. Genetic or PAD-mediated PP2A reactivation induces Jak2(V617F) inactivation/downregulation and impairs clonogenic potential of Jak2(V617F) cell lines and PV but not normal CD34(+) progenitors. Likewise, FTY720 decreases leukemic allelic burden, reduces splenomegaly, and significantly increases survival of Jak2(V617F) leukemic mice without adverse effects. Mechanistically, we show that in Jak2(V617F) cells, FTY720 antileukemic activity requires neither FTY720 phosphorylation (FTY720-P) nor SET dimerization or ceramide induction but depends on interaction with SET K209. Moreover, we show that Jak2(V617F) also utilizes an alternative sphingosine kinase-1-mediated pathway to inhibit PP2A and that FTY720-P, acting as a sphingosine-1-phosphate-receptor-1 agonist, elicits signals leading to the Jak2-PI-3Kγ-PKC-SET-mediated PP2A inhibition. Thus, PADs (eg, FTY720) represent suitable therapeutic alternatives for Jak2(V617F) MPNs.

Junctional complex and focal adhesion rearrangement mediates pulmonary endothelial barrier enhancement by FTY720 S-phosphonate.

RATIONALE: Modulation of pulmonary vascular barrier function is an important clinical goal given the devastating effects of vascular leak in acute lung injury (ALI). We previously demonstrated that FTY720 S-phosphonate (Tys), an analog of sphingosine 1-phosphate (S1P) and FTY720, has more potent pulmonary barrier protective effects than these agents in vitro and in mouse models of ALI. Tys preserves expression of the barrier-promoting S1P1 receptor (S1PR1), whereas S1P and FTY720 induce its ubiquitination and degradation. Here we further characterize the novel barrier promoting effects of Tys in cultured human pulmonary endothelial cells (EC). METHODS/RESULTS: In human lung EC, Tys significantly increased peripheral redistribution of adherens junction proteins VE-cadherin and ß-catenin and tight junction protein ZO-1. Inhibition of VE-cadherin with blocking antibody significantly attenuated Tys-induced transendothelial resistance (TER) elevation, while ZO-1 siRNA partially inhibited this elevation. Tys significantly increased focal adhesion formation and phosphorylation of focal adhesion kinase (FAK). Pharmacologic inhibition of FAK significantly attenuated Tys-induced TER elevation. Tys significantly increased phosphorylation and peripheral redistribution of the actin-binding protein, cortactin, while cortactin siRNA partially attenuated Tys-induced TER elevation. Although Tys significantly increased phosphorylation of Akt and GSK3ß, neither PI3 kinase nor GSK3ß inhibition altered Tys-induced TER elevation. Tys significantly increased Rac1 activity, while inhibition of Rac1 activity significantly attenuated Tys-induced VE-cadherin redistribution and TER elevation. CONCLUSION: Junctional complex, focal adhesion rearrangement and Rac1 activation play critical roles in Tys-mediated barrier protection in pulmonary EC. These results provide mechanistic insights into the effects of this potential ALI therapy.

Tracking sphingosine metabolism and transport in sphingolipidoses: NPC1 deficiency as a test case.

The late endosomal/lysosomal compartment (LE/LY) plays a key role in sphingolipid breakdown, with the last degradative step catalyzed by acid ceramidase. The released sphingosine can be converted to ceramide in the ER and transported by ceramide transfer protein (CERT) to the Golgi for conversion to sphingomyelin. The mechanism by which sphingosine exits LE/LY is unknown but Niemann-Pick C1 protein (NPC1) has been suggested to be involved. Here, we used sphingomyelin, ceramide and sphingosine labeled with [(3)H] in carbon-3 of the sphingosine backbone and targeted them to LE/LY in low-density lipoprotein (LDL) particles. These probes traced LE/LY sphingolipid degradation and recycling as suggested by (1) accumulation of [(3)H]-sphingomyelin-derived [(3)H]-ceramide and depletion of [(3)H]-sphingosine upon acid ceramidase depletion, and (2) accumulation of [(3)H]-sphingosine-derived [(3)H]-ceramide and attenuation of [(3)H]-sphingomyelin synthesis upon CERT depletion. NPC1 silencing did not result in the accumulation of [(3)H]-sphingosine derived from [(3)H]-sphingomyelin/LDL or [(3)H]-ceramide/LDL. Additional evidence against NPC1 playing a significant role in LE/LY sphingosine export was obtained in experiments using the [(3)H]-sphingolipids or a fluorescent sphingosine derivative in NPC1 knock-out cells. Instead, NPC1-deficient cells displayed an increased affinity for sphingosine independently of protein-mediated lipid transport. This likely contributes to the increased sphingosine content of NPC1 cells.

Organization of fluorescent cholesterol analogs in lipid bilayers - lessons from cyclodextrin extraction.

To characterize the structure and dynamics of cholesterol in membranes, fluorescent analogs of the native molecule have widely been employed. The cholesterol content in membranes is in general manipulated by using water-soluble cyclodextrins. Since the interactions between cyclodextrins and fluorescent-labeled cholesterol have not been investigated in detail so far, we have compared the cyclodextrin-mediated membrane extraction of three different fluorescent cholesterol analogs (one bearing a NBD and two bearing BODIPY moieties). Extraction of these analogs was followed by measuring the Förster resonance energy transfer between a rhodamine moiety linked to phosphatidylethanolamine and the labeled cholesterol. The extraction kinetics revealed that the analogs are differently extracted from membranes. We examined the orientation of the analogs within the membrane and their influence on lipid condensation using NMR and EPR spectroscopies. Our data indicate that the extraction of fluorescent sterols from membranes is determined by several parameters, including their impact on lipid order, their hydrophobicity, their intermolecular interactions with surrounding lipids, their orientation within the bilayer, and their affinity with the exogenous acceptor.

Synthesis of cholesterol analogues bearing BODIPY fluorophores by Suzuki or Liebeskind-Srogl cross-coupling and evaluation of their potential for visualization of cholesterol pools.

We report a synthetic route to BODIPY-cholesterol conjugates in which the key steps were Suzuki or Liebeskind-Srogl cross-coupling of cholesterol phenyl moieties with structurally diverse BODIPY scaffolds. All conjugates feature single-bonded and hydrophobic linkages between the fluorophore and sterol that are devoid of heteroatoms. Using HeLa cells, we show that these BODIPY-cholesterol analogues can be used simultaneously with the parent BODIPY-cholesterol for cell imaging and flow cytometry. The BODIPY-cholesterol analogues exhibit similar cellular localization in HeLa cells and show similar cholesterol efflux properties from THP-1 cells to HDL acceptors. These results demonstrate that the red-shifted BODIPY-cholesterol analogues behave in a manner similar to unlabeled cholesterol and are useful probes for simultaneous visualization of intracellular cholesterol pools and for monitoring cholesterol efflux from cells to extracellular acceptors.

FTY720 (s)-phosphonate preserves sphingosine 1-phosphate receptor 1 expression and exhibits superior barrier protection to FTY720 in acute lung injury.

OBJECTIVE: Effective therapies are needed to reverse the increased vascular permeability that characterizes acute inflammatory diseases such as acute lung injury. FTY720 is a pharmaceutical analog of the potent barrier-enhancing phospholipid, sphingosine 1-phosphate. Because both FTY720 and sphingosine 1-phosphate have properties that may limit their usefulness in patients with acute lung injury, alternative compounds are needed for therapeutic use. The objective of this study is to characterize the effects of FTY720 (S)-phosphonate, a novel analog of FTY720-phosphate, on variables of pulmonary vascular permeability in vitro and alveolar-capillary permeability in vivo. SETTING: University-affiliated research institute. SUBJECTS: Cultured human pulmonary endothelial cells; C57BL/6 mice. INTERVENTIONS: Endothelial cells were stimulated with sphingosine 1-phosphate receptor 1 agonists to determine effects on sphingosine 1-phosphate receptor 1 expression. Acute lung injury was induced in C57BL/6 mice with bleomycin to assess effects of sphingosine 1-phosphate receptor 1 agonists. MEASUREMENTS AND MAIN RESULTS: FTY720 (S)-phosphonate potently increases human pulmonary endothelial cell barrier function in vitro as measured by transendothelial electrical resistance. Reduction of sphingosine 1-phosphate receptor 1 with small interference RNA significantly attenuates this transendothelial electrical resistance elevation. FTY720 (S)-phosphonate maintains endothelial sphingosine 1-phosphate receptor 1 protein expression in contrast to greater than 50% reduction after incubation with sphingosine 1-phosphate, FTY720, or other sphingosine 1-phosphate receptor 1 agonists. FTY720 (S)-phosphonate does not induce ß-arrestin recruitment, sphingosine 1-phosphate receptor 1 ubiquitination, and proteosomal degradation that occur after other agonists. Intraperitoneal administration of FTY720 (S)-phosphonate every other day for 1 week in normal or bleomycin-injured mice maintains significantly higher lung sphingosine 1-phosphate receptor 1 expression compared with FTY720. FTY720 fails to protect against bleomycin-induced acute lung injury in mice, while FTY720 (S)-phosphonate significantly decreases lung leak and inflammation. CONCLUSION: FTY720 (S)-phosphonate is a promising barrier-promoting agent that effectively maintains sphingosine 1-phosphate receptor 1 levels and improves outcomes in the bleomycin model of acute lung injury.

Differential basolateral-apical distribution of scavenger receptor, class B, type I in cultured cells and the liver.

The high-density lipoprotein (HDL) receptor, scavenger receptor class B, type I (SR-BI), mediates selective cholesteryl ester uptake into the liver, which finally results in cholesterol secretion into the bile. Despite several reports, the distribution of hepatic SR-BI between the sinusoidal and canalicular membranes is still under debate. We present immunohistological data using specific markers showing that the bulk of SR-BI is present in sinusoidal membranes and, to a lesser extent, in canalicular membranes in murine and human liver sections. In addition, SR-BI was detected in preparations of rat liver canalicular membranes. We also compared the in vivo findings to HepG2 cells, a widely used in vitro hepatocyte model. Interestingly, SR-BI was enriched in bile canalicular-like (BC-like) structures in polarized HepG2 cells, which were cultivated either conventionally to form a monolayer or in Matrigel to form three-dimensional structures. Fluorescently labeled HDL was transported into close proximity of BC-like structures, whereas HDL labeled with the fluorescent cholesterol analog BODIPY-cholesterol was clearly detected within these structures. Importantly, similarly to human and mouse liver, SR-BI was localized in basolateral membranes in three-dimensional liver microtissues from primary human liver cells. Our results demonstrate that SR-BI is highly enriched in sinusoidal membranes and is also found in canalicular membranes. There was no significant basolateral-apical redistribution of hepatic SR-BI in fasting and refeeding experiments in mice. Furthermore, in vitro studies in polarized HepG2 cells showed explicit differences as SR-BI was highly enriched in BC-like structures. These structures are, however, functional and accumulated HDL-derived cholesterol. Thus, biological relevant model systems should be employed when investigating SR-BI distribution in vitro.

Membrane orientation and lateral diffusion of BODIPY-cholesterol as a function of probe structure.

Cholesterol tagged with the BODIPY fluorophore via the central difluoroboron moiety of the dye (B-Chol) is a promising probe for studying intracellular cholesterol dynamics. We synthesized a new BODIPY-cholesterol probe (B-P-Chol) with the fluorophore attached via one of its pyrrole rings to carbon-24 of cholesterol (B-P-Chol). Using two-photon fluorescence polarimetry in giant unilamellar vesicles and in the plasma membrane (PM) of living intact and actin-disrupted cells, we show that the BODIPY-groups in B-Chol and B-P-Chol are oriented perpendicular and almost parallel to the bilayer normal, respectively. B-Chol is in all three membrane systems much stronger oriented than B-P-Chol. Interestingly, we found that the lateral diffusion in the PM was two times slower for B-Chol than for B-P-Chol, although we found no difference in lateral diffusion in model membranes. Stimulated emission depletion microscopy, performed for the first time, to our knowledge, with fluorescent sterols, revealed that the difference in lateral diffusion of the BODIPY-cholesterol probes was not caused by anomalous subdiffusion, because diffusion of both analogs in the PM was free but not hindered. Our combined measurements show that the position and orientation of the BODIPY moiety in cholesterol analogs have a severe influence on lateral diffusion specifically in the PM of living cells.