Murine Peritoneal Macrophages Research Articles

Comprehensive Functional Analysis of N-Linked Glycans on Ebola Virus GP1

ABSTRACTEbola virus (EBOV) entry requires the virion surface-associated glycoprotein (GP) that is composed of a trimer of heterodimers (GP1/GP2). The GP1 subunit contains two heavily glycosylated domains, the glycan cap and the mucin-like domain (MLD). The glycan cap contains only N-linked glycans, whereas the MLD contains both N- and O-linked glycans. Site-directed mutagenesis was performed on EBOV GP1 to systematically disrupt N-linked glycan sites to gain an understanding of their role in GP structure and function. All 15 N-glycosylation sites of EBOV GP1 could be removed without compromising the expression of GP. The loss of these 15 glycosylation sites significantly enhanced pseudovirion transduction in Vero cells, which correlated with an increase in protease sensitivity. Interestingly, exposing the receptor-binding domain (RBD) by removing the glycan shield did not allow interaction with the endosomal receptor, NPC1, indicating that the glycan cap/MLD domains mask RBD residues required for binding. The effects of the loss of GP1 N-linked glycans on Ca2+-dependent (C-type) lectin (CLEC)-dependent transduction were complex, and the effect was unique for each of the CLECs tested. Surprisingly, EBOV entry into murine peritoneal macrophages was independent of GP1 N-glycans, suggesting that CLEC-GP1 N-glycan interactions are not required for entry into this important primary cell. Finally, the removal of all GP1 N-glycans outside the MLD enhanced antiserum and antibody sensitivity. In total, our results provide evidence that the conserved N-linked glycans on the EBOV GP1 core protect GP from antibody neutralization despite the negative impact the glycans have on viral entry efficiency.

HPLC-DAD analysis, antileishmanial, antiproliferative, and antibacterial activities of Lacistema pubescens: an Amazonian medicinal plant.

Species of the genus Lacistema are traditionally used by Brazilian and Peruvian indigenous communities. The present study investigated the in vitro antileishmanial activity against several Leishmania species, cytotoxicity in murine peritoneal macrophages, antiproliferative activity against HL60 and Jurkat cells, and antibacterial activities against seven bacteria strains of the aerial parts of the methanolic crude extract and fractions of Lacistema pubescens. In addition, their chemical profile was also evaluated. Hexane fraction showed the most significant IC50 values against all promastigotes of Leishmania species tested, except for L. chagasi (IC50 = 4.2 µg/mL for L. major and IC50 = 3.5 µg/mL for L. amazonensis). This fraction also exhibited a strong activity against amastigotes of L. amazonensis (IC50 = 6.9 µg/mL). The antiproliferative activity was also observed for methanolic extract and hexane fraction with IC50 = 47.2 µg/mL and IC50 = 39.7 µg/mL for HL60, respectively. Regarding the antimicrobial activity, the overall antibacterial activity was not very significative. Phytol and sitosterol were identified in the methanolic extract. Additionally, previous studies also revealed the presence of those compounds in the hexane fraction. Among other compounds, phytol and sitosterol were probably involved in the antileishmanial and cytotoxicity activities observed in this study.

Scientific validation of the Chinese caterpillar medicinal mushroom, Ophiocordyceps sinensis (Ascomycetes) from India: immunomodulatory and antioxidant activity.

In the present study, we aimed to elucidate the antioxidant property and anti-inflammatory activity of the aqueous extract of the Indian species of Ophiocordyceps sinensis (AECS), which demonstrates medicinal activity against numerous diseases. The chemical composition of AECS was quantified using a colorimeteric technique to determine the total phenolic and flavonoid contents. Antioxidant activity was determined by assays for 2,2'-azinobis(3-ethylbenzothiazoline-6-sulphonic acid)diammonium salt (ABTS); 2,2-diphenyl-1-picryl-hydrazyl (DPPH); and ferric reducing antioxidant power (FRAP). Adenosine nucleoside and nitrogenous bases (adenine and uracil) were also quantified by high-performance thin layer chromatography (HPTLC). Furthermore, the aqueous extract was also analyzed for anti-inflammatory activity in vitro using THP1 cells. THP1 cells were treated with and without lipopolysaccharide (LPS) and AECS (at 25 µg/mL, 50 µg/mL and 100 µg/mL, respectively) for 24 h. After 24 h, supernatants were harvested and kept at -80°C until the cytokine assays were performed. Furthermore, nitric oxide (NO) content was also estimated in treated and untreated murine peritoneal macrophages using Griess reagent. AECS significantly suppressed LPS-induced release of TNF-α and IL-1β in THP1 cells and significantly suppressed NO release in macrophage cells without exerting any toxic effect. These results indicate the anti-inflammatory activity of AECS. Additionally, this extract also has an antioxidant property, as high contents of phenols and flavonoids are present in the extract with considerable reducing power. The results of this study clearly demonstrate the potent antioxidant property and anti-inflammatory activity of AECS. Therefore, consumption of AECS may be clinically useful to protect against inflammatory diseases.

Extra virgin olive oil polyphenolic extracts downregulate inflammatory responses in LPS-activated murine peritoneal macrophages suppressing NFκB and MAPK signalling pathways.

Extra virgin olive oil (EVOO) is obtained from the fruit of the olive tree Olea europaea L. Phenolic compounds present in EVOO have recognized anti-oxidant and anti-inflammatory properties. However, the activity of the total phenolic fraction extracted from EVOO and the action mechanisms involved are not well defined. The present study was designed to evaluate the potential anti-inflammatory mechanisms of the polyphenolic extract (PE) from EVOO on LPS-stimulated peritoneal murine macrophages. Nitric oxide (NO) production was analyzed by the Griess method and intracellular reactive oxygen species (ROS) by fluorescence analysis. Moreover, changes in the protein expression of the pro-inflammatory enzymes, inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2 and microsomal prostaglandin E synthase-1 (mPGES-1), as well as the role of nuclear transcription factor kappa B (NFκB) and mitogen-activated protein kinase (MAPK) signalling pathways, were analyzed by Western blot. PE from EVOO reduced LPS-induced oxidative stress and inflammatory responses through decreasing NO and ROS generation. In addition, PE induced a significant down-regulation of iNOS, COX-2 and mPGES-1 protein expressions, reduced MAPK phosphorylation and prevented the nuclear NFκB translocation. This study establishes that PE from EVOO possesses anti-inflammatory activities on LPS-stimulated murine macrophages.

Cholesterol-Induced Impact on Murine Macrophage Responsiveness Involves down-Regulation of Mevalonate Pathway

Free cholesterol (Ch) and its oxidative derivatives, oxysterols, are often accumulated in macrophages during chronic inflammation and atherogenesis. The effects of Ch and oxysterols on the balance of pro- and anti-inflammatory cytokines in inflammatory response and the role of mevalonate pathway in the effects of these sterols are studied poorly. We studied the effects of cholesterol, oxysterols, atorvastatin, and mevalonic acid on the LPS-induced TNF-α, IL-10, and TGF-β1 production in macrophage cell culture. The study was carried out in murine peritoneal macrophages preincubated for 4 h with Ch (5 µg/mL), 25-hydroxycholesterol (25-OH-Ch) (5 µg/mL), 7-keto-Ch (5 µg/mL), farnesol (10 µM), or atorvastatin (5 µmol/mL) in the presence or absence of 1 mM of mevalonate. The cells were further incubated in the presence or absence of E. coli 0111:B4 lipopolysaccharide (LPS) for 24 h, and cytokine concentrations in incubation media were determined. Macrophages preincubation with Ch, 25-OH-Ch, or atorvastatin decreased LPS-induced TNF-α production in cell cultures, while supplementation of preincubation medium with mevalonic acid abrogated the effects of atorvastatin and Ch. The Ch, 25-OH-Ch, 7-keto-Ch and atorvastatin significantly reduced IL-10 production by LPS–stimulated macrophages, while farnesol had no effect. Supplementation of Ch or atorvastatin-containing preincubation medium with mevalonate restored IL-10 production. The TGF-β1 production was significantly enhanced by the presence of Ch or atorvastatin in preincubation medium as compared to the control level in non-treated macrophages, while 25-OH-Ch or farnesol decreased profoundly TGF-β1 production. Mevalonate abrogated the effect of Ch or atorvastatin but not of 25-OH-Ch or farnesol. These results allow to conclude, that Ch is able to promote anti-inflammatory and fibrogenic macrophage response, which is connected, at least in part, with the deficiency of mevalonate pathway intermediates, particularly to the deficiency of farnesol, whereas hydroxysterols produce tolerogenic, but not fibrogenic effect, independently of mevalonate pathway.

Ursolic acid protects monocytes against metabolic stress-induced priming and dysfunction by preventing the induction of Nox4

AimsDietary supplementation with ursolic acid (UA) prevents monocyte dysfunction in diabetic mice and protects mice against atherosclerosis and loss of renal function. The goal of this study was to determine the molecular mechanism by which UA prevents monocyte dysfunction induced by metabolic stress. Methods and resultsMetabolic stress sensitizes or “primes” human THP-1 monocytes and murine peritoneal macrophages to the chemoattractant MCP-1, converting these cells into a hyper-chemotactic phenotype. UA protected THP-1 monocytes and peritoneal macrophages against metabolic priming and prevented their hyper-reactivity to MCP-1. UA blocked the metabolic stress-induced increase in global protein-S-glutathionylation, a measure of cellular thiol oxidative stress, and normalized actin-S-glutathionylation. UA also restored MAPK phosphatase-1 (MKP1) protein expression and phosphatase activity, decreased by metabolic priming, and normalized p38 MAPK activation. Neither metabolic stress nor UA supplementation altered mRNA or protein levels of glutaredoxin-1, the principal enzyme responsible for the reduction of mixed disulfides between glutathione and protein thiols in these cells. However, the induction of Nox4 by metabolic stress, required for metabolic priming, was inhibited by UA in both THP-1 monocytes and peritoneal macrophages. ConclusionUA protects THP-1 monocytes against dysfunction by suppressing metabolic stress-induced Nox4 expression, thereby preventing the Nox4-dependent dysregulation of redox-sensitive processes, including actin turnover and MAPK-signaling, two key processes that control monocyte migration and adhesion. This study provides a novel mechanism for the anti-inflammatory and athero- and renoprotective properties of UA and suggests that dysfunctional blood monocytes may be primary targets of UA and related compounds.

GLP-2 Suppresses LPS-Induced Inflammation in Macrophages by Inhibiting ERK Phosphorylation and NF-κB Activation

Background/Aims: GLP-2 has been shown to exert anti-inflammatory effects, but the underlying molecular mechanisms remained undefined. As macrophages are important in the development and maintenance of inflammation, we investigated whether exogenous GLP-2 modulates the expression of pro-inflammatory proteins in LPS stimulated murine peritoneal macrophages. Methods: Macrophages were pretreated with various concentrations of GLP-2 for 1 h and then stimulated with LPS. The effects on pro-inflammatory enzymes (iNOS and COX-2), and pro-inflammatory cytokines (TNF-a, IL-1ß and IL-6) were analysed by Western blotting, ELISA and qRT-PCR. We also examined whether NF-κB or MAPK signaling was involved in the effects of GLP-2. Results: In macrophages, GLP-2 blunted the effect of LPS on protein and mRNA expression levels of iNOS, COX-2, TNF-a, IL-1ß and IL-6. Pre-incubation of macrophages with GLP-2 also blunted LPS-induced IκB-a degradation, IκB-a phosphorylation and NF-κB translocation. In the presence of GLP-2, the effect of LPS treatment on ERK phosphorylation was also profoundly blunted. GLP-2 did, however, not significantly modify the effects of LPS on p38 and JNK activities. Conclusions: These findings demonstrate that in LPS primed macrophages, GLP-2 reduced pro-inflammatory enzymes and cytokine production via mechanisms involving the suppression of NF-κB activity and ERK phosphorylation.

Overall Estimation of Anti-Oxidant Activity by Mammal Macrophage

Super oxide anion had been known as a critical role in a phagocytic cell when microorganisms invade inside. However, this radical seldom need for a clean environment such in a developed country. Moreover, this agent sometimes triggered as serious factor in the site of inflammation. There are many papers which concerned newly found anti-oxidative substances. However, many reports only focused on the molecular basis in vitro, how oxidative stress damaged the human tissue. The key factor for oxidative stress needs to be discussed first for immunological standpoint, such as armed reduction treaty. In other words, minimum armed force is necessary for an invasion by outer or inner irregular cells. On the basis of phagocytic function of the cell in vivo, we tried to access anti-oxidative activity by employing peritoneal murine macrophage and human monocyte. So, the purpose of this study is to establish a conventional but tailor made system for which is useful for evaluating the anti-oxidative activity to different constition. Especially in human peripheral monocyte, the sample was tested each constitution such in granulocyte rich type and lymphocyte rich typ. In order to establish the direct estimation by phagocytic cell, a murine peritoneal exudates cell suspension was obtained after one week of oral administration with anti-oxidative sample. The samples we selected were traditional Chinese medicine, Shi-Quan-Da-Bu-Tang (SDT), Bu-Zong-Ye-Qi-Tang (BYT) and Shao-Chin-Rong-Tang sCRT), Agaricus, Chlorella, and Propolis. The amount of generated super oxide anion was calculated through measuring the optical density change of this supernatant using cytochrome-C which was reduced by generated super oxide anion in the phagocytic cell. The generated super oxide anion after administration of Agaricus, Chlorella, SDT, BYT, sCRT and Propolis were 2.64, 1.95, 1.24, 1.25, 2.88, 2.55 × 10﹣5 mmol/ml, respectively, whereas 2.85 × 10﹣5 mmol/ml in control group. The fermented sample from SDT, BYT, sCRT and microfied Propolis were also tested and the value of super oxide anion were 0.62, 0.84, 1.50 and 2.55 × 10﹣5 mmol/ml, respectively. In this report, we discussed the direct method for measuring the generated super oxide anion of macrophages, especially from different constitution.

Neonatal murine macrophages show enhanced chemotactic capacity upon toll-like receptor stimulation

The neonatal surgical patient is threatened by exuberant inflammatory reactions. Neonatal macrophages are key players in this process. We investigated the ability of neonatal macrophages to initiate a local inflammatory reaction upon exposure to different bacterial or viral ligands to toll-like receptors (TLRs). Peritoneal wash outs from neonatal (<24 h) and adult (42 days) C57BL/6J mice were gained by peritoneal lavages. In a first set of experiments, macrophages were purified and stimulated for 6 h by four different TLR ligands. mRNA was extracted for transcriptome analysis. In a second set of experiments, lipopolysaccharide was applied into peritoneal cavities. After 6 h of incubation, the cellular composition of the inflamed cavities was evaluated by cytological staining as well as chipcytometry. Neonatal murine peritoneal macrophages differed significantly in the expression of pro- and anti-chemotactic genes. Functional assignment of these genes revealed enhanced chemotactic potential of neonatal macrophages and was confirmed by a higher influx of pro-inflammatory cells into neonatal peritoneal cavities. Neonatal peritoneal macrophages demonstrated an enhanced chemotactic potential upon stimulation with four TLR ligands. This was associated with an increased influx of inflammatory cells to the peritoneal cavity. This might contribute to the strong inflammatory responses of neonates and preterms.

Rapid induction of REDD1 gene expression in macrophages in response to stress-related catecholamines

In the present study, we examined the effect of stress-related catecholamines adrenaline and noradrenaline on macrophage expression of a new host defense factor REDD1 using murine macrophage cell line RAW264.7 and murine peritoneal macrophages. Short-term adrenaline exposure (15–60min) upregulated REDD1 mRNA expression and its protein synthesis in macrophages. This adrenaline-induced REDD1 expression was completely blocked by β2-adrenoceptor selective antagonist ICI 118,551, whereas β2-adrenoceptor specific agonist salmeterol markedly enhanced REDD1 expression. Moreover, noradrenaline increased REDD1 mRNA expression at doses higher than the effective doses of adrenaline. The effect of adrenaline on REDD1 mRNA expression was mimicked by treatment with membrane-permeable cAMP analog 8-Br-cAMP. Thus, increased intracellular cAMP level resulting from β2-adrenoceptor stimulation appeared to be responsible for adrenaline-induced REDD1 mRNA expression. However, inhibiting protein kinase A (PKA) activity had no significant effect on REDD1 mRNA expression after β2-adrenoceptor stimulation. In addition, exchange protein activated by cAMP (Epac) agonist 8-CPT-20-O-Me-cAMP had no effect on REDD1 mRNA expression. Thus, β2-adrenoceptor-mediated increase in cAMP levels seems to induce REDD1 mRNA expression in macrophages through a PKA- and Epac-independent pathway.

Complexes of different nitrogen donor heterocyclic ligands with SbCl3 and PhSbCl2 as potential antileishmanial agents against SbIII-sensitive and -resistant parasites

Novel trivalent antimony complexes with the nitrogen donor heterocyclic ligand 2,2′-bipyridine (bipy), 1,10-phenanthroline (phen) or dipyrido[3,2-d:2′,3′-f]quinoxaline (dpq) have been synthesized by the reaction with SbCl3 or PhSbCl2. The crystal structures of [Sb(phen)Cl3] and [PhSb(phen)Cl2]CH3COOH were determined and shown to adopt a distorted square pyramid geometry with a five-coordinated Sb center. Surprisingly, all the complexes, the ligands and PhSbCl2 showed very high antileishmanial activities, with IC50 in the nanomolar range against SbIII-sensitive and -resistant Leishmania infantum (syn. Leishmania chagasi) and Leishmania amazonensis strains. These compounds were much more active against these Leishmania strains than the old trivalent drug potassium antimonyl tartrate. [PhSb(phen)Cl2]CH3COOH complex was found to be the most active compound and the lack of cross-resistance of PhSbCl2 suggests that the transport pathways of this compound across the cell membrane differ from those responsible for the resistance of Leishmania to Sb(OH)3. In the case of the complexes with PhSbCl2, our data supports the model that both ligand and metal contributed to the overall activity of the complex. Furthermore, among the complexes with SbCl3, only bipy showed an improved activity upon complexation. Cytotoxicity evaluations of these compounds against murine peritoneal macrophages showed high selective indexes in the range of 7–70 for [Sb(phen)Cl3], [Sb(bipy)Cl3] and [Sb(dpq)Cl3] complexes, being much more selective than potassium antimonyl tartrate. In conclusion, this study presents a set of new antileishmanial agents including one of the most active Sb-based compounds ever reported, which can contribute to the development of new chemotherapeutic strategies against leishmaniasis including Sb-resistant cases.

Melatonin modulates microsomal PGE synthase 1 and NF‐E2‐related factor‐2‐regulated antioxidant enzyme expression in LPS‐induced murine peritoneal macrophages

Increasing evidence demonstrates that melatonin regulates inflammatory and immune processes acting as both an activator and inhibitor of these responses. Nevertheless, the molecular mechanisms of its anti-inflammatory action remain unclear. Here we have characterized the cellular mechanisms underlying the redox modulation of LPS-stimulated inflammatory responses in murine peritoneal macrophages by melatonin to provide insight into its anti-inflammatory effects. Murine peritoneal macrophages were isolated and treated with melatonin in the presence or absence of LPS (5 μg·mL(-1) ) for 18 h. Cell viability was determined using sulforhodamine B assay and NO production was measured using the Griess reaction. Pro-inflammatory enzymes and transcription factors were detected by Western blotting. Without affecting cell viability, melatonin (12.5, 25, 50 and 100 μM) reduced the level of nitrites, inducible NOS (iNOS), COX-2 and microsomal PGE synthase-1 (mPGES1) protein, and p38 MAPK phosphorylation, and prevented NF-κB translocation. Furthermore, melatonin treatment significantly increased NF-E2-related factor 2 (Nrf2) and haem oxygenase 1 (HO1) protein levels in murine macrophages exposed to LPS. Melatonin reduced pro-inflammatory mediators and enhanced the expression of HO1 via NF-κB, p38 MAPK and Nrf2 cascade signalling pathways in murine macrophages. Thus, melatonin might be a promising target for diseases associated with overactivation of macrophages.

BP5 regulated B cell development promoting anti-oxidant defence

Bursa of Fabricius is the humoral immune system for B cell differentiation and antibody production. Bursopentine (BP5) is a novel immunomodulatory peptide and significantly stimulated an antigen-specific immune response in mice. BP5 was also found to protect LPS-activated murine peritoneal macrophages from oxidative stress. In this study, the effects of BP5 on B cell development were examined. The results suggested that BP5 markedly promoted B cell development by increasing CFU-pre B, and affected the redox homeostasis regulation of B cells. To study the molecular mechanism of effect of bursal-derived BP5, this research utilized 2D-E and MALDI-TOF/TOF to analyze the differentially expressed proteins of BP5-treated WEHI-231 cells. The results showed that BP5 affected the redox homeostasis regulation of WEHI-231 cells and induced alterations in the protein expression profiles related to the oxidoreduction coenzyme metabolic process, precursor metabolites and energy, proteolysis, RNA splicing and translation and cellular process, respectively. BP5 also induced glucose-6-phosphate dehydrogenase (G6PD) activity, an essential anti-oxidant cofactor. We found that the redox homeostasis regulation effect of BP5 was reduced in G6PD-deficient cells. These data suggested that BP5 affected the redox balance toward reducing conditions by promoting the expression of G6PD, which in turn regulated the glutathione redox cycle and other processes.

Abstract 19083: Macrophage Expression of Pcsk9 Influences Atherosclerosis Development

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secretory protein that regulates low-density lipoprotein (LDL) cholesterol levels by binding and degrading hepatic LDL receptor (LDLR), thus contributing to atherosclerosis. Little is known of PCSK9 effect in macrophages and whether this contributes to the development of the atheroma. First, we demonstrated that murine peritoneal macrophages (MPM) express endogenous (m) PCSK9 both at the mRNA and protein level. Then, to test the effect of human (h) PCSK9 on atherosclerosis we developed transgenic mice expressing hPCSK9 from multiple tissues and cells, including macrophages. As it occurs in hepatocytes, hPCSK9 reduced LDLR levels in MPM. The effect on LDLR was more evident in resident than in elicited MPM, and was completely abolished after culturing MPM in medium containing FBS or LPDS, that cause LDLR upregulation. Co-immunoprecipitation experiments showed a direct interaction of hPCSK9 with LDL-R. In addition, we found that PCSK9 also interacts with LRP-1, a member of the LDL-R gene family involved in modulation of inflammatory responses and cell survival. It has been recently reported that PCSK9 reduces LRP1 levels in transduced renal HEK293T cells. However, we found that the interaction of hPCSK9 with LRP1 did not affect either total or membrane levels of the receptor. Surprisingly, analyses of spleen lysates from hPCSK9 transgenics revealed an increase in the percentage of Ly6C hi and CD11b positive cells compared with WT. To study the effect of macrophage PCSK9 in the atheroma, bone marrow from hPCSK9/apoE -/- or apoE -/- mice was transplanted into apoE -/- recipients. Low levels of hPCSK9 were detected in serum of hPCSK9/apoE -/- marrow recipients, but no effects on cholesterol were noted. Although lesion size was not different, lesion composition analyses showed a higher percentage of Ly6C hi positive cells in recipients of hPCSK9/apoE -/- marrow compared to controls (7.4±1.5% vs. 5.6±1.1%, respectively, p &lt;0.05). Our results show for first time that PCSK9 expression by macrophages directly influences atherosclerotic plaque composition in the absence of changes in serum cholesterol levels, suggesting a direct effect of macrophage PCSK9 in inflammation and plaque development.

Inducement of Cytokine Release by GFPBW2, a Novel Polysaccharide from Fruit Bodies of Grifola frondosa, through Dectin-1 in Macrophages

Polysaccharides, especially β-glucans isolated from various species of mushrooms, are considered as biological response modifiers (BRMs) to be widely used in the treatment of cancer, especially due to their immunostimulatory activity. We herein characterized the structure of a novel water-soluble homogeneous polysaccharide (GFPBW2) from the fruit bodies of mushroom Grifola frondosa and investigated its immunomodulatory activity in vitro. GFPBW2 was purified from the alkali-extracted fractions by stepwise elution with a molecular weight of 26.2 kDa. On the basis of infrared and NMR spectroscopy, methylation and monosaccharide composition analysis, partial acid hydrolysis, and Smith degradation, its structure was elucidated to possess a backbone consisting of β-d-1,3- and β-d-1,4-linked glucopyranosyl residues, with branches attached to O-6 of β-d-1,3-linked glucopyranosyl residues. Functionally, it is an effective inducer of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) secretion in murine resident peritoneal macrophages. Using quartz crystal microbalance (QCM) analysis, we found that GFPBW2 could bind dendritic cell-associated C-type lectin-1 (Dectin-1) with an affinity constant (Kd) value of 1.08 × 10 (-7) M, while it could activate Syk and enhance TNF-α production in RAW264.7 cells overexpressing wild type but not mutant Dectin-1. Furthermore, Syk, NF-κB signaling, and cytokine release in resident peritoneal macrophages induced by GFPBW2 could be significantly inhibited by a specific Dectin-1 blocking reagent, Laminarin. These data suggested that GFPBW2 might be a potential ligand of Dectin-1, and the potential of GFPBW2 to activate macrophage through triggering cytokine secretion might be attributed, at least in part, to the involvement of Dectin-1.

Long-chain fatty alcohols from evening primrose oil inhibit the inflammatory response in murine peritoneal macrophages

Ethnopharmacological relevanceEvening primrose (Oenothera biennis L., Onagraceae) is a wild medicinal plant of Central American origin that is now one of the most widely used herbal medicines in different parts of the world. Oil extracted from it seeds is traditionally used in the treatment of eczema, asthma, rheumatoid arthritis, breast problem, premenstrual and menopausal syndrome, all they have an inflammatory component. The present study demonstrates the in vitro anti-inflammatory effect of long-chain fatty alcohols, minor compounds isolated from Evening primrose oil (EPO). Material and methodsA mixture of long chain fatty alcohols (LCFAs) was isolated from the non-triacylglycerol fraction of the EPO. Hexacosanol (C26OH: 38.65%), tetracosanol (C24OH: 31.59%), docosanol (C22OH: 11.36%) and octocosanol (C28OH: 7.64%), were the major constituents, identified and quantified by GC and GC–MS. LCFA was tested with LPS stimulated murine peritoneal macrophage. This fraction, significantly and dose-dependently decreased nitric oxide production induced by LPS (P<0.001) and the inhibitory effect seems to be consequence of an action at the level of the inducible nitric-oxide synthethase (iNOS) gene enzyme expression rather than to a direct inhibitory action on enzyme activity. The release of PLA2 and TXB2 also was significantly inhibited by LCFAs (P<0.001) although LCFAs did not affect to PGE2 generation, however the western blot assay showed that LCFAs reduced cyclooxygenase-2 enzyme gene expression at all doses assayed. In the same way, the secretion of inflammatory cytokines interleukin 1β (IL-1β) and tumour necrosis factor α (TNF-α) from LPS-stimulated murine macrophage, were also significantly reduced (P<0.001). ConclusionThese results demonstrates the anti-inflammatory activity of LCFAs, providing an additional value about the role of bioactive minor compounds in the beneficial effect of EPO and supports its traditional uses in inflammatory processes management.

Preparation of Neomytilan, a Bioglycan from the Mussel Crenomytilus grayanus, and its Immunostimulating Activity

Bioglycans isolated from the Far-East mussel Crenomytilus grayanus were compared analytically. It was found that neomytilan, a bioglycan with reduced protein content, and the previously isolated mytilan stimulated phagocytosis of murine peritoneal macrophages in vitro and influenced the synthesis of pro-inflammatory cytokines TNF-α, IL-6, and IFN-γ.

Identification of a Tissue-Specific, C/EBPβ-Dependent Pathway of Differentiation for Murine Peritoneal Macrophages

Macrophages and dendritic cells (DC) are distributed throughout the body and play important roles in pathogen detection and tissue homeostasis. In tissues, resident macrophages exhibit distinct phenotypes and activities, yet the transcriptional pathways that specify tissue-specific macrophages are largely unknown. We investigated the functions and origins of two peritoneal macrophage populations in mice: small and large peritoneal macrophages (SPM and LPM, respectively). SPM and LPM differ in their ability to phagocytose apoptotic cells, as well as in the production of cytokines in response to LPS. In steady-state conditions, SPM are sustained by circulating precursors, whereas LPM are maintained independently of hematopoiesis; however, both populations are replenished by bone marrow precursors following radiation injury. Transcription factor analysis revealed that SPM and LPM express abundant CCAAT/enhancer binding protein (C/EBP)-β. Cebpb(-/-) mice exhibit elevated numbers of SPM-like cells but lack functional LPM. Alveolar macrophages are also missing in Cebpb(-/-) mice, although macrophage populations in the spleen, kidney, skin, mesenteric lymph nodes, and liver are normal. Adoptive transfer of SPM into Cebpb(-/-) mice results in SPM differentiation into LPM, yet donor SPM do not generate LPM after transfer into C/EBPβ-sufficient mice, suggesting that endogenous LPM inhibit differentiation by SPM. We conclude that C/EBPβ plays an intrinsic, tissue-restricted role in the generation of resident macrophages.

The lignan glycosides lyoniside and saracoside poison the unusual type IB topoisomerase of Leishmania donovani and kill the parasite both in vitro and in vivo

Lignans are diphenyl propanoids with vast range of biological activities. The present study provides an important insight into the anti-leishmanial activities of two lignan glycosides, viz. lyoniside and saracoside. These compounds inhibit catalytic activities of topoisomerase IB (LdTopIB) of Leishmania donovani in non-competitive manner and stabilize the LdTopIB mediated cleavage complex formation both in vitro and in Leishmania promastigotes and subsequently inhibit the religation of cleaved strand. These two compounds not only poison LdTopIB but also can interact with the free enzyme LdTopIB. We have also shown that lyoniside and saracoside are cytotoxic to promastigotes and intracellular amastigotes. The protein–DNA complex formation leads to double strand breaks in DNA which ultimately triggers apoptosis-like cell death in the parasite. Along with their cytotoxicity towards sodium antimony gluconate (SAG) sensitive AG83 strain, their ability to kill SAG resistant GE1 strain makes these two compounds potential anti-leishmanial candidates. Not only they effectively kill L. donovani amastigotes inside macrophages in vitro, lyoniside and saracoside demonstrated strong anti-leishmanial efficacies in BALB/c mice model of leishmaniasis. Treatment with these lignan glycosides produce nitric oxide and reactive oxygen species which result in almost complete clearance of the liver and splenic parasite burden. These compounds do not inhibit human topoisomerase IB upto 200μM concentrations and had poor cytotoxic effect on uninfected cultured murine peritoneal macrophages upto 100μM concentrations. Taken together it can be concluded that these compounds can be developed into excellent therapeutic agent against deadly disease leishmaniasis.

Responses of Murine and Human Macrophages to Leptospiral Infection: A Study Using Comparative Array Analysis

Leptospirosis is a re-emerging tropical infectious disease caused by pathogenic Leptospira spp. The different host innate immune responses are partially related to the different severities of leptospirosis. In this study, we employed transcriptomics and cytokine arrays to comparatively calculate the responses of murine peritoneal macrophages (MPMs) and human peripheral blood monocytes (HBMs) to leptospiral infection. We uncovered a series of different expression profiles of these two immune cells. The percentages of regulated genes in several biological processes of MPMs, such as antigen processing and presentation, membrane potential regulation, and the innate immune response, etc., were much greater than those of HBMs (>2-fold). In MPMs and HBMs, the caspase-8 and Fas-associated protein with death domain (FADD)-like apoptosis regulator genes were significantly up-regulated, which supported previous results that the caspase-8 and caspase-3 pathways play an important role in macrophage apoptosis during leptospiral infection. In addition, the key component of the complement pathway, C3, was only up-regulated in MPMs. Furthermore, several cytokines, e.g. interleukin 10 (IL-10) and tumor necrosis factor alpha (TNF-alpha), were differentially expressed at both mRNA and protein levels in MPMs and HBMs. Some of the differential expressions were proved to be pathogenic Leptospira-specific regulations at mRNA level or protein level. Though it is still unclear why some animals are resistant and others are susceptible to leptospiral infection, this comparative study based on transcriptomics and cytokine arrays partially uncovered the differences of murine resistance and human susceptibility to leptospirosis. Taken together, these findings will facilitate further molecular studies on the innate immune response to leptospiral infection.