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1.  Cytokine and Nitric Oxide Levels in Patients with Sepsis – Temporal Evolvement and Relation to Platelet Mitochondrial Respiratory Function 
PLoS ONE  2014;9(5):e97673.
Background
The levels of nitric oxide (NO) and various cytokines are known to be increased during sepsis. These signaling molecules could potentially act as regulators and underlie the enhancement of mitochondrial function described in the later phase of sepsis. Therefore, we investigated the correlation between observed changes in platelet mitochondrial respiration and a set of pro- and anti-inflammatory cytokines as well as NO plasma levels in patients with sepsis.
Methods and Results
Platelet mitochondrial respiration and levels of TNFα, MCP-1 (monocyte chemotactic protein-1), INFγ (interferon-γ), IL-1β, IL-4, IL-5, IL-6, IL-8, IL-10 and IL-17 and NO were analyzed in 38 patients with severe sepsis or septic shock at three time points during one week following admission to the ICU. Citrate synthase, mitochondrial DNA and cytochrome c were measured as markers of cellular mitochondrial content. All mitochondrial respiratory states increased over the week analyzed (p<0.001). IL-8 levels correlated with maximal mitochondrial respiration on day 6–7 (p = 0.02, r2 = 0.22) and was also higher in non-survivors compared to survivors on day 3–4 and day 6–7 (p = 0.03 respectively). Neither NO nor any of the other cytokines measured correlated with respiration or mortality. Cytochrome c levels were decreased at day 1–2 by 24±5% (p = 0.03) and returned towards values of the controls at the last two time points. Citrate synthase activity and mitochondrial DNA levels were similar to controls and remained constant throughout the week.
Conclusions
Out of ten analyzed cytokines and nitric oxide, IL-8 correlated with the observed increase in mitochondrial respiration. This suggests that cytokines as well as NO do not play a prominent role in the regulation of platelet mitochondrial respiration in sepsis. Further, the respiratory increase was not accompanied by an increase in markers of mitochondrial content, suggesting a possible role for post-translational enhancement of mitochondrial respiration rather than augmented mitochondrial mass.
doi:10.1371/journal.pone.0097673
PMCID: PMC4020920  PMID: 24828117
2.  Critical Role of TLR2 and MyD88 for Functional Response of Macrophages to a Group IIA-Secreted Phospholipase A2 from Snake Venom 
PLoS ONE  2014;9(4):e93741.
The snake venom MT-III is a group IIA secreted phospholipase A2 (sPLA2) enzyme with functional and structural similarities with mammalian pro-inflammatory sPLA2s of the same group. Previously, we demonstrated that MT-III directly activates the innate inflammatory response of macrophages, including release of inflammatory mediators and formation of lipid droplets (LDs). However, the mechanisms coordinating these processes remain unclear. In the present study, by using TLR2−/− or MyD88−/− or C57BL/6 (WT) male mice, we report that TLR2 and MyD88 signaling have a critical role in MT-III-induced inflammatory response in macrophages. MT-III caused a marked release of PGE2, PGD2, PGJ2, IL-1β and IL-10 and increased the number of LDs in WT macrophages. In MT-III-stimulated TLR2−/− macrophages, formation of LDs and release of eicosanoids and cytokines were abrogated. In MyD88−/− macrophages, MT-III-induced release of PGE2, IL-1β and IL-10 was abrogated, but release of PGD2 and PGJ2 was maintained. In addition, COX-2 protein expression seen in MT-III-stimulated WT macrophages was abolished in both TLR2−/− and MyD88−/− cells, while perilipin 2 expression was abolished only in MyD88−/− cells. We further demonstrated a reduction of saturated, monounsaturated and polyunsaturated fatty acids and a release of the TLR2 agonists palmitic and oleic acid from MT-III-stimulated WT macrophages compared with WT control cells, thus suggesting these fatty acids as major messengers for MT-III-induced engagement of TLR2/MyD88 signaling. Collectively, our findings identify for the first time a TLR2 and MyD88-dependent mechanism that underlies group IIA sPLA2-induced inflammatory response in macrophages.
doi:10.1371/journal.pone.0093741
PMCID: PMC3981733  PMID: 24718259
3.  Dengue Induces Platelet Activation, Mitochondrial Dysfunction and Cell Death through Mechanisms that Involve DC-SIGN and Caspases 
Background
Dengue is the most prevalent human arbovirus disease in the world. Dengue infection may cause a range of clinical manifestation from self-limiting febrile illness through life-threatening syndrome accompanied by bleeding and shock. Thrombocytopenia is frequently observed in mild and severe disease, however the mechanisms involved in DENV-induced platelet activation and thrombocytopenia are incompletely understood.
Patients/ Methods
Freshly-isolated platelets from patients with dengue were evaluated for markers of activation, mitochondrial alterations and activation of cell death pathways. In parallel, we determined whether DENV induced direct activation and apoptosis of platelets that were obtained from healthy subjects.
Results
We found that platelets from DENV-infected patients display increased activation when compared to control subjects. Moreover, platelets from DENV-infected patients exhibited classic signs of the intrinsic pathway of apoptosis that include increased surface phosphatidylserine exposure, mitochondrial depolarization and activation of caspase-9 and 3. Indeed, thrombocytopenia was shown to strongly associate with enhanced platelet activation and cell death in DENV-infected patients. Platelet activation, mitochondrial dysfunction and caspase-dependent phosphatidylserine exposure on platelets were also observed when platelets from healthy subjects were directly exposed to DENV in vitro. DENV-induced platelet activation was shown to occur through mechanisms largely dependent of DC-SIGN.
Conclusions
Together our results demonstrate that platelets from patients with dengue present signs of activation, mitochondrial dysfunction, and activation of apoptosis caspase cascade, which may contribute to the genesis of thrombocytopenia in patients with dengue. Our results also suggest the involvement of DC-SIGN as a critical receptor in DENV-dependent platelet activation.
doi:10.1111/jth.12178
PMCID: PMC3971842  PMID: 23433144
apoptosis; DC-SIGN; dengue; mitochondrial dysfunction; platelet activation; thrombocytopenia
4.  A Fluorescence-Based Assay for Measuring the Redox Potential of 5-Lipoxygenase Inhibitors 
PLoS ONE  2014;9(2):e87708.
The activities and side effects of 5-lipoxygenase (5-LO) inhibitors can be predicted by identifying their redox mechanisms. In this study, we developed a fluorescence-based method to measure the redox potential of 5-LO inhibitors and compared it to the conventional, absorbance-based method. After the pseudo-peroxidase reaction, the amount of remaining lipid peroxide was quantified using the H2DCFDA (2′,7′-dichlorodihydrofluorescein diacetate) fluorescence dye. Our method showed large signal windows and provided comparable redox potential values. Importantly, the redox mechanisms of known inhibitors were accurately measured with the fluorescence assay, whereas the conventional, absorbance-based method showed contradictory results. Our findings suggest that our developed method is a better alternative for classifying the redox potential of 5-LO inhibitors, and the fluorescence assay can be effectively used to study the mechanisms of action that are related to redox cycling.
doi:10.1371/journal.pone.0087708
PMCID: PMC3912022  PMID: 24498359
5.  Mycobacteria Bypass Mucosal NF-kB Signalling to Induce an Epithelial Anti-Inflammatory IL-22 and IL-10 Response 
PLoS ONE  2014;9(1):e86466.
The mechanisms by which mycobacteria subvert the inflammatory defence to establish chronic infection remain an unresolved question in the pathogenesis of tuberculosis. Using primary epithelial cells, we have analysed mycobacteria induced epithelial signalling pathways from activation of TLRs to cytokine secretion. Mycobacterium bovis bacilli Calmette-Guerin induced phosphorylation of glycogen synthase kinase (GSK)3 by PI3K–Akt in the signalling pathway downstream of TLR2 and TLR4. Mycobacteria did not supress NF-κB by activating the peroxisome proliferator-activated receptor γ. Instead the pro-inflammatory NF-κB was bypassed by mycobacteria induced GSK3 inhibition that promoted the anti-inflammatory transcription factor CREB. Mycobacterial infection did not thus induce mucosal pro-inflammatory response as measured by TNFα and IFNγ secretion, but led to an anti-inflammatory IL-10 and IL-22 production. Apart from CREB, MAP3Ks p38 and ERK1/2 activated the transcription factor AP-1 leading to IL-6 production. Interestingly, blocking of TLR4 before infection decreased epithelial IL-6 secretion, but increased the CREB-activated IL-10 production. Our data indicate that mycobacteria supress epithelial pro-inflammatory production by supressing NF-κB activation thereby shifting the infection towards an anti-inflammatory state. This balance between the host immune response and the pathogen could determine the outcome of infection.
doi:10.1371/journal.pone.0086466
PMCID: PMC3904915  PMID: 24489729
8.  Lysophosphatidylcholine Triggers TLR2- and TLR4-Mediated Signaling Pathways but Counteracts LPS-Induced NO Synthesis in Peritoneal Macrophages by Inhibiting NF-κB Translocation and MAPK/ERK Phosphorylation 
PLoS ONE  2013;8(9):e76233.
Background
Lysophosphatidylcholine (LPC) is the main phospholipid component of oxidized low-density lipoprotein (oxLDL) and is usually noted as a marker of several human diseases, such as atherosclerosis, cancer and diabetes. Some studies suggest that oxLDL modulates Toll-like receptor (TLR) signaling. However, effector molecules that are present in oxLDL particles and can trigger TLR signaling are not yet clear. LPC was previously described as an attenuator of sepsis and as an immune suppressor. In the present study, we have evaluated the role of LPC as a dual modulator of the TLR-mediated signaling pathway.
Methodology/Principal Findings
HEK 293A cells were transfected with TLR expression constructs and stimulated with LPC molecules with different fatty acid chain lengths and saturation levels. All LPC molecules activated both TLR4 and TLR2-1 signaling, as evaluated by NF-қB activation and IL-8 production. These data were confirmed by Western blot analysis of NF-қB translocation in isolated nuclei of peritoneal murine macrophages. However, LPC counteracted the TLR4 signaling induced by LPS. In this case, NF-қB translocation, nitric oxide (NO) synthesis and the expression of inducible nitric oxide synthase (iNOS) were blocked. Moreover, LPC activated the MAP Kinases p38 and JNK, but not ERK, in murine macrophages. Interestingly, LPC blocked LPS-induced ERK activation in peritoneal macrophages but not in TLR-transfected cells.
Conclusions/Significance
The above results indicate that LPC is a dual-activity ligand molecule. It is able to trigger a classical proinflammatory phenotype by activating TLR4- and TLR2-1-mediated signaling. However, in the presence of classical TLR ligands, LPC counteracts some of the TLR-mediated intracellular responses, ultimately inducing an anti-inflammatory phenotype; LPC may thus play a role in the regulation of cell immune responses and disease progression.
doi:10.1371/journal.pone.0076233
PMCID: PMC3848743  PMID: 24312681
9.  Bacterial Clearance Is Improved in Septic Mice by Platelet-Activating Factor-Acetylhydrolase (PAF-AH) Administration 
PLoS ONE  2013;8(9):e74567.
Current evidence indicates that dysregulation of the host inflammatory response to infectious agents is central to the mortality of patients with sepsis. Strategies to block inflammatory mediators such as PAF have been investigated as adjuvant therapies for sepsis. PAF-AH, the enzyme responsible for PAF degradation, showed positive results in pre-clinical studies and phase II clinical trials, but the results of a phase III study were disappointing. In this study, we investigated the potential protective mechanism of PAF-AH in sepsis using the murine model of cecal ligation and puncture (CLP). Treatment with rPAF-AH increased peritoneal fluid levels of the anti-inflammatory mediators MCP-1/CCL2 after CLP. The numbers of bacteria (CFU) in the peritoneal cavity were decreased in the rPAF-AH-treated group, indicating more efficient bacterial clearance after rPAF-AH treatment. Interestingly, we observed increased levels of nitric oxide (NO) after PAF-AH administration, and rPAF-AH treatment did not decrease CFU numbers either in iNOS-deficient mice or in CCR2-deficient mice. We concluded that administration of exogenous rPAF-AH reduced inflammatory injury, altered cytokine levels and favored bacterial clearance with a clear impact on mortality through modulation of MCP-1/CCL2 and NO levels in a clinically relevant sepsis model.
doi:10.1371/journal.pone.0074567
PMCID: PMC3771912  PMID: 24069320
10.  Differential Regulation of Cysteinyl Leukotriene Receptor Signaling by Protein Kinase C in Human Mast Cells 
PLoS ONE  2013;8(8):e71536.
Cysteinyl leukotrienes (cys-LTs) are a group of lipid mediators that are potent bronchoconstrictors, powerful inducers of vascular leakage and potentiators of airway hyperresponsiveness. Cys-LTs play an essential role in asthma and are synthesized as well as activated in mast cells (MCs). Cys-LTs relay their effects mainly through two known GPCRs, CysLT1R and CysLT2R. Although protein kinase C (PKC) isoforms are implicated in the regulation of CysLT1R function, neither the role of PKCs in cys-LT-dependent MC inflammatory signaling nor the involvement of specific isoforms in MC function are known. Here, we show that PKC inhibition augmented LTD4 and LTE4-induced calcium influx through CysLT1R in MCs. In contrast, inhibition of PKCs suppressed c-fos expression as well MIP1β generation by cys-LTs. Interestingly, cys-LTs activated both PKCα and PKCε isoforms in MC. However, knockdown of PKCα augmented cys-LT mediated calcium flux, while knockdown of PKCε attenuated cys-LT induced c-fos expression and MIP1β generation. Taken together, these results demonstrate for the first time that cys-LT signaling downstream of CysLT1R in MCs is differentially regulated by two distinct PKCs which modulate inflammatory signals that have significant pathobiologic implications in allergic reactions and asthma pathology.
doi:10.1371/journal.pone.0071536
PMCID: PMC3744564  PMID: 23977066
11.  Adiponectin Inhibits Neutrophil Phagocytosis of Escherichia coli by Inhibition of PKB and ERK 1/2 MAPK Signalling and Mac-1 Activation 
PLoS ONE  2013;8(7):e69108.
Full length adiponectin is a potent immune modulatory adipokine, impacting upon the actions of several immune cells. Neutrophil oxidative burst has been shown to decrease in response to adiponectin, and we speculated that it could have other effects on neutrophil function. Here we report that adiponectin reduces the phagocytic ability of human neutrophils, decreasing significantly the ingestion of opsonised E. coli by these cells in whole blood (p<0.05) and as isolated neutrophils (p<0.05). We then determined the mechanisms involved. We observed that the activation of Mac-1, the receptor engaged in complement-mediated phagocytosis, was decreased by adiponectin in response to E. coli stimulation. Moreover, treatment of neutrophils with adiponectin prior to incubation with E. coli significantly inhibited signalling through the PI3K/PKB and ERK 1/2 pathways, with a parallel reduction of F-actin content. Studies with pharmacological inhibitors showed that inhibition of PI3K/PKB, but not ERK 1/2 signalling was able to prevent the activation of Mac-1. In conclusion, we propose that adiponectin negatively affects neutrophil phagocytosis, reducing the uptake of E. coli and inhibiting Mac-1 activation, the latter by blockade of the PI3K/PKB signal pathway.
doi:10.1371/journal.pone.0069108
PMCID: PMC3723777  PMID: 23935932
12.  Mechanisms of Siglec-F-Induced Eosinophil Apoptosis: A Role for Caspases but Not for SHP-1, Src Kinases, NADPH Oxidase or Reactive Oxygen 
PLoS ONE  2013;8(6):e68143.
Background
Siglec-F and Siglec-8 are functional paralog proapoptotic cell surface receptors expressed on mouse and human eosinophils, respectively. Whereas Siglec-8 mediated death involves caspases and/or reactive oxygen species (ROS) generation and mitochondrial injury, very little is known about Siglec-F-mediated signaling and apoptosis. Therefore the objective of the current experiments was to better define apoptosis pathways mediated by Siglec-F and Siglec-8. Given that Siglec-F-induced apoptosis is much less robust than Siglec-8-induced apoptosis, we hypothesized that mechanisms involved in cell death via these receptors would differ.
Methods
Consequences of engagement of Siglec-F on mouse eosinophils were studied by measuring ROS production, and by performing apoptosis assays using eosinophils from normal, hypereosinophilic, NADPH oxidase-deficient, src homology domain-containing protein tyrosine phosphatase (SHP)-1-deficient, and Lyn kinase-deficient mice. Inhibitors of caspase and Src family kinase activity were also used.
Results
Engagement of Siglec-F induced mouse eosinophil apoptosis that was modest in magnitude and dependent on caspase activity. There was no detectable ROS generation, or any role for ROS, NADPH oxidase, SHP-1, or Src family kinases in this apoptotic process.
Conclusions
These data suggest that Siglec-F-mediated apoptosis is different in both magnitude and mechanisms when compared to published data on Siglec-8-mediated human eosinophil apoptosis. One likely implication of this work is that models targeting Siglec-F in vivo in mice may not provide identical mechanistic predictions for consequences of Siglec-8 targeting in vivo in humans.
doi:10.1371/journal.pone.0068143
PMCID: PMC3695997  PMID: 23840825
13.  Identifying Intracellular Sites of Eicosanoid Lipid Mediator Synthesis with EicosaCell Assays 
Eicosanoids, arachidonic acid-derived signaling lipid mediators, are newly formed and nonstorable molecules that have important roles in physiological and pathological processes. EicosaCell is a microscopic assay that enables the intracellular detection and localization of eicosanoid lipid mediator-synthesizing compartments by means of a strategy to covalently cross-link and immobilize eicosanoids at their sites of synthesis followed by immunofluorescent-based localization of the targeted eicosanoid. EicosaCell is a versatile assay which allows analyses of different types of cell preparations, such as cells isolated from humans or harvested cells from in vivo models of inflammation and adherent or suspension cells stimulated in vitro. EicosaCell assays have been successfully used to identify different intracellular compartments of synthesis of prostaglandins and leukotrienes upon cellular activation. This is of particular interest given that over the past decade intracellular compartmentalization of eicosanoid-synthetic machinery has emerged both as a key component in the regulation of eicosanoid synthesis and in delineating functional intracellular and extracellular actions of eicosanoids. This review covers basics of EicosaCell assay including its selection of reagents, immunodetection design as well as some troubleshooting recommendations.
doi:10.1007/978-1-61779-024-9_16
PMCID: PMC3679532  PMID: 21370037
Eicosanoids; EicosaCell; Immunofluorescence detection; Bioactive lipid mediators; Heterobifunctional cross-linker
14.  EicosaCell – An Immunofluorescent-Based Assay to Localize Newly Synthesized Eicosanoid Lipid Mediators at Intracellular Sites 
Eicosanoids (prostaglandins, leukotrienes and lipoxins) are a family of signaling lipids derived from arachidonic acid that have important roles in physiological and pathological processes. Over the past years, it has been established that successful eicosanoid production is not merely determined by arachidonic acid and eicosanoid-forming enzymes availability, but requires sequential interactions between specific biosynthetic proteins acting in cascade and may involve very unique spatial interactions. Direct assessment of specific subcellular locales of eicosanoid synthesis has been elusive, as those lipid mediators are newly formed, not stored and often rapidly released upon cell stimulation. In this chapter, we discuss the EicosaCell protocol for intracellular detection of eicosanoid-synthesizing compartments by means of a strategy to covalently cross-link and immobilize the lipid mediators at their sites of synthesis followed by immunofluorescent-based localization of the targeted eicosanoid.
doi:10.1007/978-1-60761-950-5_10
PMCID: PMC3679533  PMID: 21153792
Eicosanoids; prostaglandin; leukotriene; biosynthesis; compartmentalization; carbodiimide; EDAC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide); lipid droplets; phagosomes; perinuclear
15.  Acyl Chain-Dependent Effect of Lysophosphatidylcholine on Endothelium-Dependent Vasorelaxation 
PLoS ONE  2013;8(5):e65155.
Previously we identified palmitoyl-, oleoyl-, linoleoyl-, and arachidonoyl-lysophosphatidylcholine (LPC 16:0, 18:1, 18:2 and 20:4) as the most prominent LPC species generated by endothelial lipase (EL). In the present study, we examined the impact of those LPC on acetylcholine (ACh)- induced vascular relaxation. All tested LPC attenuated ACh-induced relaxation, measured ex vivo, using mouse aortic rings and wire myography. The rank order of potency was as follows: 18:2>20:4>16:0>18:1. The attenuating effect of LPC 16:0 on relaxation was augmented by indomethacin-mediated cyclooxygenase (COX)-inhibition and CAY10441, a prostacyclin (PGI2)- receptor (IP) antagonist. Relaxation attenuated by LPC 20:4 and 18:2 was improved by indomethacin and SQ29548, a thromboxane A2 (TXA2)- receptor antagonist. The effect of LPC 20:4 could also be improved by TXA2- and PGI2-synthase inhibitors. As determined by EIA assays, the tested LPC promoted secretion of PGI2, TXA2, PGF2α, and PGE2, however, with markedly different potencies. LPC 16:0 was the most potent inducer of superoxide anion production by mouse aortic rings, followed by LPC 18:2, 20:4 and 18:1, respectively. The strong antioxidant tempol recovered relaxation impairment caused by LPC 18:2, 18:1 and 20:4, but not by LPC 16:0. The tested LPC attenuate ACh-induced relaxation through induction of proconstricting prostanoids and superoxide anions. The potency of attenuating relaxation and the relative contribution of underlying mechanisms are strongly related to LPC acyl-chain length and degree of saturation.
doi:10.1371/journal.pone.0065155
PMCID: PMC3669280  PMID: 23741477
16.  Imaging Lipid Bodies Within Leukocytes with Different Light Microscopy Techniques 
Lipid bodies, also known as lipid droplets, are present in most eukaryotic cells. In leukocytes, lipid bodies are functionally active organelles with central roles in inflammation and are considered structural markers of inflammatory cells in a range of diseases. The identification of lipid bodies has methodological limitations because lipid bodies dissipate upon drying or dissolve upon fixation and staining with alcohol-based reagents. Here we discuss several techniques to detect and visualize lipid bodies within leukocytes by light microscopy. These techniques include staining with osmium or use of different fluorescent probes such as Nile red, BODIPY, Oil red, P96 and immunofluorescence labeling for adipose differentiation-related protein (ADRP).
doi:10.1007/978-1-60761-950-5_9
PMCID: PMC3659330  PMID: 21153791
Lipid bodies; lipid droplets; leukocytes; bright field and fluorescence microscopy; osmium staining; nile red; oil red O; BODIPY; 1-pyrenedodecanoic acid; adipose differentiation-related protein (ADRP)
17.  A Novel Small Compound SH-2251 Suppresses Th2 Cell-Dependent Airway Inflammation through Selective Modulation of Chromatin Status at the Il5 Gene Locus 
PLoS ONE  2013;8(4):e61785.
IL-5 is a key cytokine that plays an important role in the development of pathological conditions in allergic inflammation. Identifying strategies to inhibit IL-5 production is important in order to establish new therapies for treating allergic inflammation. We found that SH-2251, a novel thioamide-related small compound, selectively inhibits the differentiation of IL-5-producing Th2 cells. SH-2251 inhibited the induction of active histone marks at the Il5 gene locus during Th2 cell differentiation. The recruitment of RNA polymerase II, and following expression of the Th2 cell-specific intergenic transcripts around the Il5 gene locus was also inhibited. Furthermore, Th2 cell-dependent airway inflammation in mice was suppressed by the oral administration of SH-2251. Gfi1, a transcriptional repressor, was identified as a downstream target molecule of SH-2251 using a DNA microarray analysis. The Gfi1 expression dramatically decreased in SH-2251-treated Th2 cells, and the SH-2251-mediated inhibition of IL-5-producing Th2 cell differentiation was restored by transduction of Gfi1. Therefore, our study unearthed SH-2251 as a novel therapeutic candidate for allergic inflammation that selectively inhibits active histone marks at the Il5 gene locus.
doi:10.1371/journal.pone.0061785
PMCID: PMC3628909  PMID: 23613936
18.  Characterisation of P2Y12 Receptor Responsiveness to Cysteinyl Leukotrienes 
PLoS ONE  2013;8(3):e58305.
Leukotriene E4 (LTE4), the most stable of the cysteinyl leukotrienes (cysLTs), binds poorly to classical type 1 and 2 cysLT receptors although in asthmatic individuals it may potently induce bronchial constriction, airway hyperresponsiveness and inflammatory cell influx to the lung. A recent study has suggested that the purinergic receptor P2Y12 is required for LTE4 mediated pulmonary inflammation in a mouse model of asthma and signals in response to cysLTs. The aim of the study was to characterise the responsiveness of human P2Y12 to cysteinyl leukotrienes. Models of human CysLT1, CysLT2 and P2Y12 overexpressed in HEK293, CHO cells and human platelets were used and responsiveness to different agonists was measured using intracellular calcium, cAMP and β-arrestin recruitment assays. CysLTs induced concentration dependent calcium mobilisation in cells overexpressing CysLT1 and CysLT2 but failed to induce any calcium response in cells expressing P2Y12 or P2Y12+ Gα16. In contrast, selective P2Y12 agonists ADP and 2-MeS-ADP induced specific calcium flux in cells expressing P2Y12+ Gα16. Similarly, specific response to 2-MeS-ADP, but not to cysLTs was also observed in cells expressing P2Y12 when intracellular cAMP and β-arrestin signalling were analysed. Platelets were used as a model of human primary cells expressing P2Y12 to analyse potential signalling and cell activation through P2Y12 receptor or receptor heterodimers but no specific LTE4 responses were observed. These results show that LTE4 as well as other cysLTs do not activate intracellular signalling acting through P2Y12 and suggest that another LTE4 specific receptor has yet to be identified.
doi:10.1371/journal.pone.0058305
PMCID: PMC3589271  PMID: 23472176
19.  Proresolution Lipid Mediators in Multiple Sclerosis — Differential, Disease Severity-Dependent Synthesis — A Clinical Pilot Trial 
PLoS ONE  2013;8(2):e55859.
Background
The severity and longevity of inflammation is controlled by endogenous counter-regulatory signals. Among them are long-chain polyunsaturated fatty acid (PUFA)-derived lipid mediators, which promote the resolution of inflammation, an active process for returning to tissue homeostasis.
Objective
To determine whether endogenous production of lipid-derived resolution agonists is regulated differentially in patients with highly active and less active multiple sclerosis (MS).
Design
Matched-pairs study in University hospital Neurology department.
Patients
Based on clinical (relapse frequency) and paraclinical (MRI lesions, contrast enhancement) criteria, 10 pairs of age- and sex-matched patients with relapsing-remitting MS were assigned either to a group with highly active or less active MS. Lipid mediators were quantified in serum and cerebrospinal fluid using LC-MS/MS-based lipidomics.
Results
Levels of the key arachidonic (ω-6) and docosahexaenoic acid (ω-6)-derived mediators prostaglandins (PG), leukotrienes, hydroxyeicosatetraenoic acids (HETE) and resolution agonists lipoxin A4 (LXA4), resolvin D1 (RvD1) and neuroprotectin D1 (NPD1) were quantified. In the patient group with highly active MS, 15-HETE and PGE2 were increased, which are products of the 15-lipoxygenase and cyclooxygenase pathways. The proresolution mediator RvD1 was significantly upregulated and NPD1 was detected in the highly active group only. LXA4 levels were not increased in patients with highly active MS.
Conclusions
Lipid mediator pathways are regulated differentially in the cerebrospinal fluid of MS patients, depending on disease severity. Non-exhaustive or possibly ‘delayed’ resolution pathways may suggest a defective resolution program in patients with highly active MS. Longitudinal analyses are required to hetero-typify this differential resolution capacity, which may be associated with disease progression, longevity and eventual termination.
doi:10.1371/journal.pone.0055859
PMCID: PMC3568070  PMID: 23409068
20.  Regulation of Serotonin-Induced Trafficking and Migration of Eosinophils 
PLoS ONE  2013;8(1):e54840.
Association of the neurotransmitter serotonin (5-HT) with the pathogenesis of allergic asthma is well recognized and its role as a chemoattractant for eosinophils (Eos) in vitro and in vivo has been previously demonstrated. Here we have examined the regulation of 5-HT-induced human and murine Eos trafficking and migration at a cellular and molecular level. Eos from allergic donors and bone marrow-derived murine Eos (BM-Eos) were found to predominantly express the 5-HT2A receptor. Exposure to 5-HT or 2,5-dimethoxy-4-iodoamphetamine (DOI), a 5-HT2A/C selective agonist, induced rolling of human Eos and AML14.3D10 human Eos-like cells on vascular cell adhesion molecule (VCAM)-1 under conditions of flow in vitro coupled with distinct cytoskeletal and cell shape changes as well as phosphorylation of MAPK. Blockade of 5-HT2A or of ROCK MAPK, PI3K, PKC and calmodulin, but not Gαi-proteins, with specific inhibitors inhibited DOI-induced rolling, actin polymerization and changes in morphology of VCAM-1-adherent AML14.3D10 cells. More extensive studies with murine BM-Eos demonstrated the role of 5-HT in promoting rolling in vivo within inflamed post-capillary venules of the mouse cremaster microcirculation and confirmed that down-stream signaling of 5-HT2A activation involves ROCK, MAPK, PI3K, PKC and calmodulin similar to AML14.3D10 cells. DOI-induced migration of BM-Eos is also dependent on these signaling molecules and requires Ca2+. Further, activation of 5-HT2A with DOI led to an increase in intracellular Ca2+ levels in murine BM-Eos. Overall, these data demonstrate that 5-HT (or DOI)/5-HT2A interaction regulates Eos trafficking and migration by promoting actin polymerization associated with changes in cell shape/morphology that favor cellular trafficking and recruitment via activation of specific intracellular signaling molecules (ROCK, MAPK, PI3K and the PKC-calmodulin pathway).
doi:10.1371/journal.pone.0054840
PMCID: PMC3553162  PMID: 23372779
21.  Statins Decrease Neuroinflammation and Prevent Cognitive Impairment after Cerebral Malaria 
PLoS Pathogens  2012;8(12):e1003099.
Cerebral malaria (CM) is the most severe manifestation of Plasmodium falciparum infection in children and non-immune adults. Previous work has documented a persistent cognitive impairment in children who survive an episode of CM that is mimicked in animal models of the disease. Potential therapeutic interventions for this complication have not been investigated, and are urgently needed. HMG-CoA reductase inhibitors (statins) are widely prescribed for cardiovascular diseases. In addition to their effects on the inhibition of cholesterol synthesis, statins have pleiotropic immunomodulatory activities. Here we tested if statins would prevent cognitive impairment in a murine model of cerebral malaria. Six days after infection with Plasmodium berghei ANKA (PbA) mice displayed clear signs of CM and were treated with chloroquine, or chloroquine and lovastatin. Intravital examination of pial vessels of infected animals demonstrated a decrease in functional capillary density and an increase in rolling and adhesion of leukocytes to inflamed endothelium that were reversed by treatment with lovastatin. In addition, oedema, ICAM-1, and CD11b mRNA levels were reduced in lovastatin-treated PbA-infected mice brains. Moreover, HMOX-1 mRNA levels are enhanced in lovastatin-treated healthy and infected brains. Oxidative stress and key inflammatory chemokines and cytokines were reduced to non-infected control levels in animals treated with lovastatin. Fifteen days post-infection cognitive dysfunction was detected by a battery of cognition tests in animals rescued from CM by chloroquine treatment. In contrast, it was absent in animals treated with lovastatin and chloroquine. The outcome was similar in experimental bacterial sepsis, suggesting that statins have neuroprotective effects in severe infectious syndromes in addition to CM. Statin treatment prevents neuroinflammation and blood brain barrier dysfunction in experimental CM and related conditions that are associated with cognitive sequelae, and may be a valuable adjuvant therapeutic agent for prevention of cognitive impairment in patients surviving an episode of CM.
Author Summary
Cerebral malaria (CM) is the direst consequence of Plasmodium falciparum infection. Cognitive impairment is a common sequela in children surviving CM. Identification of adjunctive therapies that reduce the complications of CM in survivors is a priority. Statins have been suggested for the treatment of neuroinflammatory disorders due to their pleiotropic effects. Here, we examined the effects of lovastatin on neuroinflammation in experimental CM, and its effect on the prevention of cognitive impairment. Lovastatin reduced adhesion and rolling of leukocytes in brain vessels, inhibited blood-brain barrier disruption, and reversed decreases in cerebral capillary density. Lovastatin also inhibited ICAM-1 and CD11b mRNA expression while increasing HMOX-1 mRNA levels. Proinflammatory cytokines and markers of oxidative stress were lower in the brains of infected mice treated with lovastatin. Lovastatin administered together with antimalarial drugs during the acute phase of the disease-protected survivors from impairment in both contextual and aversive memory 15 days after infection. Similar results were observed in a model of bacterial sepsis. Our findings support the possibility that statins may be valuable pharmacologic tools in treatment of patients with neuroinflammation associated with severe systemic inflammatory syndromes. Clinical trials with statins in CM and sepsis should be speedily considered to examine this point.
doi:10.1371/journal.ppat.1003099
PMCID: PMC3531520  PMID: 23300448
22.  Effects of PPARγ and RBP4 Gene Variants on Metabolic Syndrome in HIV-Infected Patients with Anti-Retroviral Therapy 
PLoS ONE  2012;7(11):e49102.
Background
PPARγ and RBP4 are known to regulate lipid and glucose metabolism and insulin resistance. The influences of PPARγ (C1431T and Pro12Ala) and RBP4 (−803GA) polymorphisms on metabolic syndrome in HIV-infected patients receiving anti-retroviral therapy were examined in this study.
Materials and Methods
A cross-sectional study of HIV-1 infected adults with antiretroviral therapy for more than one year in the National Cheng Kung University Hospital was conducted. The gene polymorphisms were determined by quantitative PCR.
Results
Ninety-one patients were included in the study. Eighty-two (90.1%) patients were males with a mean age of 44.4 years. For the C1431T polymorphism in PPARγ, while patients with the T allele (48.4%) had trends toward lower rate of hypertriglyceridemia, the borderline significance together with insignificant power did not support the protective effect of the T allele against development of hypertriglyceridemia. For the Pro12Ala polymorphism in PPARγ, although patients with the Pro/Ala genotype (8.8%) had a higher level of serum LDL (138.0 vs. 111.5 mg/dl, P = 0.04) and trends toward higher rates of hypercholesterolemia and serum LDL>110 mg/dl, these variables were found to be independent of the Pro/Ala genotype in the multivariate analysis. For the −803GA polymorphism in RBP4, patients with the A allele (23.1%) more often had insulin resistance (HOMA>3.8; 33.3 vs. 8.7%, P = 0.01) and more often received anti-hypoglycemic drugs (14.3 vs. 1.4%, P = 0.04). The detrimental effect of the A allele in RBP4 −803GA polymorphism on development of insulin resistance was supported by the multivariate analysis adjusting for covariates.
Conclusion
The impacts of PPARγ C1431T and Pro12Ala polymorphisms on metabolism in HIV-infected patients are not significant. RBP4 −803GA polymorphism has increased risk of insulin resistance in HIV-infected patients with anti-retroviral therapy.
doi:10.1371/journal.pone.0049102
PMCID: PMC3492303  PMID: 23145084
23.  Transcription of Liver X Receptor Is Down-Regulated by 15-Deoxy-Δ12,14-Prostaglandin J2 through Oxidative Stress in Human Neutrophils 
PLoS ONE  2012;7(10):e42195.
Liver X receptors (LXRs) are ligand-activated transcription factors of the nuclear receptor superfamily. They play important roles in controlling cholesterol homeostasis and as regulators of inflammatory gene expression and innate immunity, by blunting the induction of classical pro-inflammatory genes. However, opposite data have also been reported on the consequences of LXR activation by oxysterols, resulting in the specific production of potent pro-inflammatory cytokines and reactive oxygen species (ROS). The effect of the inflammatory state on the expression of LXRs has not been studied in human cells, and constitutes the main aim of the present work. Our data show that when human neutrophils are triggered with synthetic ligands, the synthesis of LXRα mRNA became activated together with transcription of the LXR target genes ABCA1, ABCG1 and SREBP1c. An inflammatory mediator, 15-deoxy-Δ12,14-prostaglandin J2 (15dPGJ2), hindered T0901317-promoted induction of LXRα mRNA expression together with transcription of its target genes in both neutrophils and human macrophages. This down-regulatory effect was dependent on the release of reactive oxygen species elicited by 15dPGJ2, since it was enhanced by pro-oxidant treatment and reversed by antioxidants, and was also mediated by ERK1/2 activation. Present data also support that the 15dPGJ2-induced serine phosphorylation of the LXRα molecule is mediated by ERK1/2. These results allow to postulate that down-regulation of LXR cellular levels by pro-inflammatory stimuli might be involved in the development of different vascular diseases, such as atherosclerosis.
doi:10.1371/journal.pone.0042195
PMCID: PMC3480349  PMID: 23115616
24.  Sepsis-Induced Acute Lung Injury (ALI) Is Milder in Diabetic Rats and Correlates with Impaired NFkB Activation 
PLoS ONE  2012;7(9):e44987.
Acute lung injury (ALI) develops in response to a direct insult to the lung or secondarily to a systemic inflammatory response, such as sepsis. There is clinical evidence that the incidence and severity of ALI induced by direct insult are lower in diabetics. In the present study we investigated whether the same occurs in ALI secondarily to sepsis and the molecular mechanisms involved. Diabetes was induced in male Wistar rats by alloxan and sepsis by caecal ligation and puncture surgery (CLP). Six hours later, the lungs were examined for oedema and cell infiltration in bronchoalveolar lavage. Alveolar macrophages (AMs) were cultured in vitro for analysis of IκB and p65 subunit of NFκB phosphorylation and MyD88 and SOCS-1 mRNA. Diabetic rats were more susceptible to sepsis than non-diabetics. In non-diabetic rats, the lung presented oedema, leukocyte infiltration and increased COX2 expression. In diabetic rats these inflammatory events were significantly less intense. To understand why diabetic rats despite being more susceptible to sepsis develop milder ALI, we examined the NFκB activation in AMs of animals with sepsis. Whereas in non-diabetic rats the phosphorylation of IκB and p65 subunit occurred after 6 h of sepsis induction, this did not occur in diabetics. Moreover, in AMs from diabetic rats the expression of MyD88 mRNA was lower and that of SOCS-1 mRNA was increased compared with AMs from non-diabetic rats. These results show that ALI secondary to sepsis is milder in diabetic rats and this correlates with impaired activation of NFκB, increased SOCS-1 and decreased MyD88 mRNA.
doi:10.1371/journal.pone.0044987
PMCID: PMC3443211  PMID: 23024779
25.  Myelin-Derived Lipids Modulate Macrophage Activity by Liver X Receptor Activation 
PLoS ONE  2012;7(9):e44998.
Multiple sclerosis is a chronic, inflammatory, demyelinating disease of the central nervous system in which macrophages and microglia play a central role. Foamy macrophages and microglia, containing degenerated myelin, are abundantly found in active multiple sclerosis lesions. Recent studies have described an altered macrophage phenotype after myelin internalization. However, it is unclear by which mechanisms myelin affects the phenotype of macrophages and how this phenotype can influence lesion progression. Here we demonstrate, by using genome wide gene expression analysis, that myelin-phagocytosing macrophages have an enhanced expression of genes involved in migration, phagocytosis and inflammation. Interestingly, myelin internalization also induced the expression of genes involved in liver-X-receptor signaling and cholesterol efflux. In vitro validation shows that myelin-phagocytosing macrophages indeed have an increased capacity to dispose intracellular cholesterol. In addition, myelin suppresses the secretion of the pro-inflammatory mediator IL-6 by macrophages, which was mediated by activation of liver-X-receptor β. Our data show that myelin modulates the phenotype of macrophages by nuclear receptor activation, which may subsequently affect lesion progression in demyelinating diseases such as multiple sclerosis.
doi:10.1371/journal.pone.0044998
PMCID: PMC3440367  PMID: 22984598

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