J774 Murine Macrophages Research Articles

THE USE OF NITRIC OXIDE FREE RADICAL SENSITIVE FLUOROPHORES TO DETECT MACROPHAGE PHAGOLYSOSOME ACTIVITY

Macrophages are phagocytes which facilitate innate immunity via phagocytosis, averting antagonistic effects resulting from bacterial infections. This is a strictly choreographed event initiated by bacterial-macrophage interactions between pathogen associated molecular patterns and toll-like receptors in macrophages. Consequently, the pathogen is ingested by the macrophage through a vacuole which matures to obtain an arsenal of antimicrobial properties including nitric oxide free-radicals (NO∙). Inducible nitric oxide synthase is an enzyme accountable for NO∙ production upon stimulation. This study utilized opportunistic pathogens Staphylococcus epidermidis, Serratia marcescens, an assemblage composed of the two species and a Lipopolysaccharide positive control to challenge the murine-macrophage J774 Cell-line. Phagolysosome activity was assessed using NO∙ sensitive fluorophore, DAF-FMDA. Fluorescence activity was measured for 300 seconds using a Nikon Eclipse TE200 fluorescence microscope and DXM1200F camera. In all treatments, maximal fluorophore activity was attained within 20 seconds; level of fluorophore activity was dependent on the treatment. S. epidermidis and the bacterial assemblage initiated relatively high activities (RFU = 73.48 ± 3.52 SD; RFU = 56.66 ± 4.74 respectively), comparable to the positive control (RFU = 71.66 ± 0.90). S. marcescens induction of fluorophore activity occurred, but to a lesser extent (RFU = 48.72 ± 3.36), over 20 seconds. The current study suggests the Gram positive S. epidermidis incites relatively high levels of NO∙ synthesis similar to the positive control which was primed with commercial Lipopolysaccharide in challenged macrophages while the NO∙ levels induced by Gram negative S. marcescens were inferior. The response to the bacterial assemblage largely mimicked the response to S. epidermidis alone suggesting macrophages preferentially phagocytosed this species. This study suggests that immune responses by macrophages depend on the bacterial species and therefore does not follow a consistent pattern, particularly in short term NO∙ synthesis.

Short-chain fatty acids increase expression and secretion of stromal cell-derived factor-1 in mouse and human pre-adipocytes.

Stromal cell-derived factor-1 (SDF-1) is expressed in pre-adipocytes but its role is unknown. We investigated butyrate (a histone deacetylase inhibitor--HDACi) and other short-chain fatty acids (SCFA) in the regulation of SDF-1. We further investigated whether effects of SCFA were signalled through G protein-coupled receptors FFA2 and FFA3. SDF-1 mRNA expression and protein secretion were studied in 3T3-L1 cells and human pre-adipocytes. SDF-1 was abundant, with mRNA and protein levels increased by butyrate. This was replicated with acetate and propionate, but not with trichostatin or valproate. Trichostatin inhibited SDF-1 secretion. Pertussis toxin blocked stimulation by butyrate. The order of potency of SCFA in stimulating SDF-1 (C3 > C4 > C2) is consistent with action through FFA3. Silencing the FFA3 gene abolished butyrate-stimulated SDF-1 expression and secretion. FFA3 was expressed in both pre-adipocytes and adipocytes, while FFA2 was expressed in adipocytes only. SDF-1 expression was low in murine macrophage J774.2 cells, while the SDF-1 receptor CXCR4 was absent from 3T3-L1 cells but abundant in J774.2 macrophages. In human pre-adipocytes, FFA3 was also expressed and SCFA increased SDF-1 secretion. SDF-1 and CXCR4 may mediate the interaction between adipose stromal cells and macrophages. Effects of SCFA are mediated through FFA3, but not histone deacetylase inhibition.

STAT3 as a Key Factor in Tumor Microenvironment and Cancer Stem Cell

Background Recent studies revealed that tumor-associated macrophages (TAMs) play a decisive role in the regulation of tumor progression by manipulating tumor oncogenesis, angiogenesis and immune functions within tumor microenvironments. Signal transducer and activator of transcription 3 (STAT3), which is a point of convergence for numerous oncogenic signalling pathways, is constitutively activated both in tumor cells and in immune cells in the tumor microenvironment. TAMs serve as the major components of niche microenvironments regulating cancer stem cell functions. Activation of the Stat3 in TAM caused to cancer stem cell-specific fashion trigger tumorigenesis and anticancer drug resistance in tumor. The main aim of this study is evaluation of stat3 gene expression in tumor microenvironment macrophages. Methods In this study, murine macrophage J774 A.1 cell line was used as a typical mouse macrophage for evaluation of stat3 gene expression in mRNA level. Culture condition followed by DMEM supplemented with 10% fetal bovine serum. Total RNA was extracted and converted to cDNA. PCR was performed by stat3 primers and then gene expression was evaluated by gel electrophoresis. Beta-actin was used as an internal control. Results Gel electrophoresis data was shown stat3 expression in murine J774 A.1 macrophage cell line. Conclusion Previous studies have been shown the ablation of the Stat3 gene in Immune cells in the tumor microenvironment caused to cancer stem cell inactivation. As regards stat3 was expressed in J774 A.1 cell line thus it seems that this cell line can be used for knockdown studies in order to cancer stem cell inactivation. Key words: Cancer Stem Cell, M2 Macrophages,STAT3.

Increased oxidative stress enhances endocannabinoid tone (671.13)

Cardiovascular disease (CVD) has been characterized as a chronic inflammatory disease that has become prevalent in industrialized societies. NADPH oxidase contributes to atherosclerosis through the activation of macrophages leading to the internalization of oxidized low‐density lipoproteins (oxLDL). Chronic inflammation is caused in part by monocytes invading the vasculature leading to the formation of lipid‐laden macrophage foam cells with increased flux of oxygen/nitrogen radicals and subsequent chemical modification of extracellular LDL. Endogenous cannabinoids (eCB), such as 2‐arachidonoylglycerol (2‐AG), may be a link between oxidative stress and atherosclerosis. We hypothesize that 2‐AG biosynthesis is enhanced following CD36 ligation by oxLDL and subsequent activation of diacylglycerol lipase β (DAGLβ), the key biosynthetic enzyme of 2‐AG, via upregulated NADPH oxidase activity. Treatment of murine J774 macrophages and human THP1 macrophages/monocytes with either extracellular xanthine/xanthine oxidase or phorbol 12‐myristate 13‐acetate (PMA) caused an increase in superoxide (O2•−) levels and enhanced 2‐AG biosynthesis (2.3‐fold and 2.8‐fold, respectively) compared to vehicle controls. These treatments were not cytotoxic. These data suggest a significant positive correlation between oxygen radical flux and 2‐AG biosynthesis in macrophages. Increased 2‐AG biosynthesis may be an adaptive response to elevated oxidative stress because of antioxidant and anti‐inflammatory actions associated with this bioactive lipid. Therefore, the pathogenesis of atherosclerosis within the vessel wall intima may be reduced by enhancing eCB tone.Grant Funding Source: Supported in part by NIH R15ES015348‐02

Suspension culture of Besnoitia caprae by murine macrophage.

Besnoitia caprae is a tissue cyst-forming protozoan that infects goats and has considerable economic importance in certain regions of Asia and Africa. Murine macrophage J774 cell line was inoculated with tachyzoites of Besnoitia caprae (BC-Pars isolate) collected from mice. A significant growth of tachyzoites was observed in J774. Mice were inoculated with tachyzoites harvested from J774 cell culture. Skin samples from the mice infected with tachyzoites of BC-Pars were PCR positive. One mouse showed alopecia and skin lesions on 45 DPI. Dermal lesions started from around right eye and gradually developed more and more. After euthanasia on 60 DPI, histopathological evaluation of skins around the eye showed necrosis of the epidermis and follicular adnexa with chronic inflammatory cell infiltration. Histopathological sections of their skin showed the presence of necrosis and mononuclear cell infiltration. To the authors' knowledge, this is the first report of successful production of Besnoitia caprae tachyzoites was achieved in vitro by suspension culture technique. Another interesting finding is the report of the alopecia and skin lesions around the eye in mouse that quite similar to lesions of goats due to infection of Besnoitia caprae.

Pyroglutamated Apelin-13 Inhibits Lipopolysaccharide-Induced Production of Pro-Inflammatory Cytokines in Murine Macrophage J774.1 Cells

Apelin, recently identified as an endogenous ligand of the orphan G protein-coupled receptor APJ, has multiple pathophysiological properties. In the present study, we investigated whether pyroglutamated apelin-13 ([Pyr1]-apelin-13), the most highly active isoform among the mature apelin peptide family, modulates the effect of bacterial lipopolysaccharide (LPS) on cytokine induction in a murine macrophage-like cell line, J774.1 cells. J774.1 cells expressed the APJ protein in a stationary state, and the expression of APJ was not affected by LPS stimulation. No significant effect of [Pyr1]-apelin-13 treatment alone was observed on the proliferation or cytokine production of J774.1 cells in the stationary state. However, prior to LPS stimulation, pretreatment with [Pyr1]apelin-13 for 16 h significantly diminished mRNA expression and protein secretion of inflammatory cytokine interleukin-6, which was confirmed by RT-PCR and ELISA, respectively. Western blot analysis revealed that the phosphorylation of p38 mitogen-activated protein kinase and c-Jun N-terminal kinase, but not extracellular signal-regulated kinase, which was induced by LPS, significantly decreased in [Pyr1]-apelin-13-pretreated J774.1 cells compared with untreated cells. These observations suggest that [Pyr1]-apelin-13 functions as a negative regulator of LPS-mediated pro-inflammatory responses in macrophages.

Candida albicans Triggers NLRP3-Mediated Pyroptosis in Macrophages

Pyroptosis is an inflammasome-mediated programmed cell death pathway triggered in macrophages by a variety of stimuli, including intracellular bacterial pathogens. Activation of pyroptosis leads to the secretion of interleukin-1β (IL-1β) and pore-mediated cell lysis. Although not considered an intracellular pathogen, Candida albicans is able to kill and, thereby, escape from macrophages. Here, we show that C. albicans-infected bone marrow-derived macrophages (BMDM) and murine J774 macrophages undergo pyroptotic cell death that is suppressed by glycine and pharmacologic inhibition of caspase-1. Infection of BMDM harvested from mice lacking components of the inflammasome revealed that pyroptosis was dependent on caspase-1, ASC, and NLRP3 and independent of NLRC4. In contrast to its role during intracellular bacterial infection, pyroptosis does not restrict C. albicans replication. Nonfilamentous Candida spp. did not trigger pyroptosis, while Candida krusei, which forms pseudohyphae in macrophages, triggered much lower levels than did C. albicans. Interestingly, a Saccharomyces cerevisiae strain from the filamentous background Σ1278 also triggered low, but significant, levels of pyroptosis. We have found that deletion of the transcription factor UPC2 decreases pyroptosis but has little effect on filamentation in the macrophage. In addition, a gain-of-function mutant of UPC2 induces higher levels of pyroptosis than does a matched control strain. Taken together, these data are most consistent with a model in which filamentation is necessary but not sufficient to trigger NLRP3 inflammasome-mediated pyroptosis. This is the first example of a fungal pathogen triggering pyroptosis and indicates that C. albicans-mediated macrophage damage is not solely due to hypha-induced physical disruption of cellular integrity.

Inhaled Solid Lipid Microparticles to target alveolar macrophages for tuberculosis

The goal of the work was to evaluate an anti-tubercular strategy based on breathable Solid Lipid Microparticles (SLM) to target alveolar macrophages and to increase the effectiveness of the conventional tuberculosis (TB) therapy. Rifampicin loaded SLM composed of stearic acid and sodium taurocholate were characterized for aerodynamic diameter, surface charge, physical state of the components, drug loading and release as well as drug biological activity on Bacillus subtilis strain. Moreover, SLM cytotoxicity and cell internalization ability were evaluated on murine macrophages J774 cell lines by MTT test, cytofluorimetry and confocal laser microscopy. SLM exhibited aerodynamic diameter proper to be transported up to the alveolar epithelium, negative charged surface able to promote uptake by the macrophages and preserved drug antimicrobial activity. The negligible in vitro release of rifampicin indicated the capacity of the microparticle matrix to entrap the drug preventing its spreading over the lung fluid. In vitro studies on J774 cell lines demonstrated SLM non-cytotoxicity and ability to be taken up by cell cytoplasm. The microparticulate carrier, showing features suitable for the inhaled therapy and for inducing endocytosis by alveolar macrophages, could be considered promising in a perspective of an efficacious TB inhaled therapy by means of a Dry Powder Inhaler device.

A new flavone sulfonic acid from Phyllanthus urinaria

One new flavone derivative, urinariaflavone (1), and eight known compounds (2–9) were isolated from the methanolic extract of Phyllanthus urinaria L. leaves. Their structures were established by MS, 1D and 2D NMR experiments, and comparison with literature data. All isolated compounds were tested for their cytotoxic activity against CHO (Chinese hamster ovary) and J774 (Murine macrophage) cell lines. Cytotoxic effects (IC50s∼6.00–41.30μM) on both cell lines were observed for compounds 4, 5, and 7.

Antileishmanial and cytotoxic activities of three plants used in colombian folk medicine

As part of our research on medicinal plants traditionally used in folk medicine in Casanare (Colombia), some important species were selected for further studies through phytochemical exploration. In this study, we investigate the phytochemical profile, antileishmanial and cytotoxic activities of three selected plants (Bowdichia virgilioides, Jacaranda obtusifolia, and Hymenaea courbaril). These plants are commonly used in the above-mentioned region to treat different infectious diseases. Leaves from test plants were collected in Casanare, Colombia, dried, grounded and macerated with ethanol. The chemical profile was determined by UFLC-DAD analysis and by measuring total phenolic (TP) and total flavonoid (TF) contents. They were also tested against promastigotes of Leishmania panamensis, J774 murine macrophages and THP-1 monocytes in order to examine a preliminary extracts' ability and security in a screening platform. The TP and TF contents (> 30 mg gallic acid equiv/g DE and > 3 mg quercetin equiv/g DE, respectively) were found to be reasonable. Chemical characterization of the ethanol extracts was achieved by UFLC-DAD employing a validated method for profiling herbal extracts using standards. All extracts were found to be enriched in flavonoid-related metabolites. Antileishmanial activity of extracts exhibited relatively high EC50 values (20 – 100 µg/mL). The extract from Hymenaea courbaril was the most active (EC50= 20.1 µg/mL). Additionally, a low cytotoxiciy against J774 and THP-1 was determined for all extracts (IC50 > 600 µg/mL, selectivity indexes (SI) > 8). Although the activity against promastigotes is quite low, and tests with the more relevant amastigotes must follow, these results might be related to the traditional use of these plants to treat infections. Therefore, the evaluation of test extracts against amastigotes and a bioguided fractionation to obtain bioactive compounds is currently in progress.

Virola plants from colombian amazon: LC-DAD and LC-MS-based chemical profiling and antileishmanial and cytotoxic activities

Members of genus Virola are well-known by Amazonian native people due to their medicinal properties. Virola is one of the most important genera of the Myristicaceae family. In Colombian Amazon, twenty one species can be found. We selected to investigate the LC-MS-based chemical profile and antileishmanial activity of three taxa (V. carinata, V. elongata and V. peruviana). Leaves, wood and bark of test Virola plants were collected in Guaviare, Colombia, dried, pulverized and subjected to maceration process with ethanol. Phytochemical profile was determined by measuring total phenolic and total flavonoid and by LC-DAD and LC-MS analysis of the resulting nine extracts for comparative drives by chemometrics through principal component analysis (PCA). In order to explore the effectiveness and security of the extracts as parasitocide agents, extracts were then tested against extracellular Leishmania panamensis parasites (promastigotes) and two cell lines (J774 murine macrophages and THP-1 monocytes), respectively. High contents of total phenolics (> 20 mg gallic acid equivalents/g DE) and total flavonoids (> 5 mg quercetin equivalents/g DE) were found. In addition, different chromatographic techniques resulted in the isolation of a novel flavonoid from the ethanolic extract of V. carinata, whose chemical structure was elucidated by spectroscopic methods. The chemical characterization of the other ethanolic extracts was achieved with LC-DAD and LC-MS techniques, using the isolated compound from V. carinata as standard. All ethanolic extracts were very rich in flavone-like compounds, indicating that this kind of phytoconstituents is possibly responsible for the extracts' antileishmanial activity (EC50 < 50 µg/mL). Additionally, extracts exhibited no cytotoxic activity against J774 and THP-1 cell lines (IC50 > 500 µg/mL) and therefore showed excellent selectivity indexes (SI > 5). PCA score plots revealed good correlations between chemical profile and biological activity.

UFLC-ESI-MS-based profiling, cytotoxicity and antileishmanial activity of Rhodostemonodaphne crenaticupula

Rhodostemonodaphne is a neotropical genus of lauroid shrubs and trees. In Colombia there are ten species. As part of our research on Lauraceae plants, the present study is directed to the phytochemical exploration, and cytotoxicity and antileishmanial evaluation of Rhodostemonodaphne crenaticupula Madriñan. Leaves, wood and bark of this plant were collected in Boyacá, Colombia. Plant material was separately dried, grounded and macerated with ethanol. The chemical profile was determined by UFLC-ESI-MS analyses of the obtaining extracts. They were also tested against promastigotes of Leishmania panamensis, J774 murine macrophages and THP-1 monocytes in order to examine the extracts' ability and security. Different chromatographic techniques resulted in the isolation of six aporphine alkaloids from the ethanolic extract from leaves, whose chemical structures were elucidated by spectroscopic methods. The chemical characterization of the remaining ethanolic extracts was achieved with UFLC-ESI-MS techniques, using the isolated compounds as standards. All ethanolic extracts were very rich in aporphine-related compounds, indicating that these kind of metabolites might be the bioactive compounds in the antileishmanial activity (EC50= 5 – 50 µg/mL). In order to support this, isolated alkaloids are currently being evaluated against parasites. Bark extract from R. crenaticupula was found to be the most potent antileishmanial. Additionally, extracts exhibited low cytotoxicity against J774 and THP-1 cell lines (IC50= 100 – 200 µg/mL) and showed good selectivity indexes (SI > 5). A bioguided fractionation leading to obtain the bioactive aporphine alkaloids is currently in process.

Conjugation, characterization and toxicity of lipophosphoglycan-polyacrylic acid conjugate for vaccination against leishmaniasis

Research on the conjugates of synthetic polyelectrolytes with antigenic molecules, such as proteins, peptides, or carbohydrates, is an attractive area due to their highly immunogenic character in comparison to classical adjuvants. For example, polyacrylic acid (PAA) is a weak polyelectrolyte and has been used in several biomedical applications such as immunological studies, drug delivery, and enzyme immobilization. However, to our knowledge, there are no studies that document immune-stimulant properties of PAA in Leishmania infection. Therefore, we aimed to develop a potential vaccine candidate against leishmaniasis by covalently conjugating PAA with an immunologically vital molecule of lipophosphoglycan (LPG) found in Leishmania parasites. In the study, LPG and PAA were conjugated by a multi-step procedure, and final products were analyzed with GPC and MALDI-TOF MS techniques. In cytotoxicity experiments, LPG-PAA conjugates did not indicate toxic effects on L929 and J774 murine macrophage cells. We assume that LPG-PAA conjugate can be a potential vaccine candidate, and will be immunologically characterized in further studies to prove its potential.

The loss of virulence of histone H1 overexpressing Leishmania donovani parasites is directly associated with a reduction of HSP83 rate of translation

Overexpression of Leishmania histone H1 (LeishH1) was previously found to cause a promastigote-to-amastigote differentiation handicap, deregulation of cell-cycle progression, and loss of parasite infectivity. The aim of this study was to identify changes in the proteome of LeishH1 overexpressing parasites associated with the avirulent phenotype observed. 2D-gel electrophoresis analysis revealed only a small protein subset of differentially expressed proteins in the LeishH1 overexpressing promastigotes. Among these was the chaperone HSP83, known for its protective role in Leishmania drug-induced apoptosis, which displayed lower translational rates. To investigate if the lower expression levels of HSP83 are associated with the differentiation handicap, we assayed the thermostability of parasites by subjecting them to heat-shock (25°C→37°C), a natural stress-factor occurring during stage differentiation. Heat-shock promoted apoptosis to a greater extent in the LeishH1 overexpressing parasites. Interestingly, these parasites were not only more sensitive to heat-shock but also to drug-induced [Sb(III)] cell-death. In addition, the restoration of HSP83 levels re-established drug resistance, and restored infectivity to LeishH1 overexpressing parasites in the murine J774 macrophage model. Overall, this study suggests that LeishH1 levels are critical for the parasite's stress-induced adaptation within the mammalian host, and highlights the cross-talk between pathways involved in drug resistance, apoptosis and virulence.

Potential Mechanism of the Anti-trypanosomal Activity of Organoruthenium Complexes with Bioactive Thiosemicarbazones

In the search for new metal-based drugs against diseases produced by trypanosomatid parasites, four organoruthenium(II) compounds [Ru2(p-cymene)2(L)2]X2, where L are bioactive 5-nitrofuryl-containing thiosemicarbazones and X = Cl or PF6, had been previously obtained. These compounds had shown activity on Trypanosoma brucei, the etiological agent of African trypanosomiasis. Because of genomic similarities between trypanosomatides, these ruthenium compounds were evaluated, in the current work, on Trypanosoma cruzi, the parasite responsible of American trypanosomiasis (Chagas disease). Two of them showed significant in vitro growth inhibition activity against the infective trypomastigote form of T. cruzi (Dm28c clone, IC50 = 11.69 and 59.42μM for [Ru2(p-cymene)2(L4)2]Cl2 and [Ru2(p-cymene)2(L1)2]Cl2, respectively, where HL4 = 5-nitrofuryl-N-phenylthiosemicarbazone and HL1 = 5-nitrofurylthiosemicarbazone), showing fairly good selectivities toward trypanosomes with respect to mammalian cells (J774 murine macrophages). Moreover, [Ru2(p-cymene)2(L2)2]Cl2, where HL2 = 5-nitrofuryl-N-methylthiosemicarbazone, was synthesized in order to evaluate the effect of improved solubility on biological behavior. This new chloride salt showed higher activity against T. cruzi than that of the previously synthesized hexafluorophosphate one (Dm28c clone, IC50 = 14.30μM for the former and 231.3μM for the latter). In addition, the mode of antitrypanosomal action of the organoruthenium compounds was investigated. The complexes were not only able to generate toxic free radicals through bioreduction but they also interacted with two further potential parasite targets: DNA and cruzipain, a cysteine protease which plays a fundamental role in the biological cycle of these parasites. The results suggest a "multi-target" mechanism of trypanosomicidal action for the obtained complexes.

Optimization of cell receptor-specific targeting through multivalent surface decoration of polymeric nanocarriers

Treatment of tuberculosis is impaired by poor drug bioavailability, systemic side effects, patient non-compliance, and pathogen resistance to existing therapies. The mannose receptor (MR) is known to be involved in the recognition and internalization of Mycobacterium tuberculosis. We present a new assembly process to produce nanocarriers with variable surface densities of mannose targeting ligands in a single step, using kinetically-controlled, block copolymer-directed assembly. Nanocarrier association with murine macrophage J774 cells expressing the MR is examined as a function of incubation time and temperature, nanocarrier size, dose, and PEG corona properties. Amphiphilic diblock copolymers are prepared with terminal hydroxyl, methoxy, or mannoside functionality and incorporated into nanocarrier formulations at specific ratios by Flash NanoPrecipitation. Association of nanocarriers protected by a hydroxyl-terminated PEG corona with J774 cells is size dependent, while nanocarriers with methoxy-terminated PEG coronas do not associate with cells, regardless of size. Specific targeting of the MR is investigated using nanocarriers having 0–75% mannoside-terminated PEG chains in the PEG corona. This is a wider range of mannose densities than has been previously studied. Maximum nanocarrier association is attained with 9% mannoside-terminated PEG chains, increasing uptake more than 3-fold compared to non-targeted nanocarriers with a 5kgmol−1 methoxy-terminated PEG corona. While a 5kgmol−1 methoxy-terminated PEG corona prevents non-specific uptake, a 1.8kgmol−1 methoxy-terminated PEG corona does not sufficiently protect the nanocarriers from nonspecific association. There is continuous uptake of MR-targeted nanocarriers at 37°C, but a saturation of association at 4°C. The majority of targeted nanocarriers associated with J774E cells are internalized at 37°C and uptake is receptor-dependent, diminishing with competitive inhibition by dextran. This characterization of nanocarrier uptake and targeting provides promise for optimizing drug delivery to macrophages for TB treatment and establishes a general route for optimizing targeted formulations of nanocarriers for specific delivery at targeted sites.

P2X7 Receptor Activation Induces Reactive Oxygen Species Formation and Cell Death in Murine EOC13 Microglia

The P2X7 purinergic receptor is a ligand-gated cation channel expressed on leukocytes including microglia. This study aimed to determine if P2X7 activation induces the uptake of organic cations, reactive oxygen species (ROS) formation, and death in the murine microglial EOC13 cell line. Using the murine macrophage J774 cell line as a positive control, RT-PCR, immunoblotting, and immunolabelling established the presence of P2X7 in EOC13 cells. A cytofluorometric assay demonstrated that the P2X7 agonists adenosine-5′-triphosphate (ATP) and 2′(3′)-O-(4-benzoylbenzoyl) ATP induced ethidium+ or YO-PRO-12+ uptake into both cell lines. ATP induced ethidium+ uptake into EOC13 cells in a concentration-dependent manner, with an EC50 of ~130 μM. The P2X7 antagonists Brilliant Blue G, A438079, AZ10606120, and AZ11645373 inhibited ATP-induced cation uptake into EOC13 cells by 75–100%. A cytofluorometric assay demonstrated that P2X7 activation induced ROS formation in EOC13 cells, via a mechanism independent of Ca2+ influx and K+ efflux. Cytofluorometric measurements of Annexin-V binding and 7AAD uptake demonstrated that P2X7 activation induced EOC13 cell death. The ROS scavenger N-acetyl-L-cysteine impaired both P2X7-induced EOC13 ROS formation and cell death, suggesting that ROS mediate P2X7-induced EOC13 death. In conclusion, P2X7 activation induces the uptake of organic cations, ROS formation, and death in EOC13 microglia.

Further evidence for the anti-inflammatory activity of oleocanthal: Inhibition of MIP-1α and IL-6 in J774 macrophages and in ATDC5 chondrocytes

AimsGiven the relevance of degenerative joint diseases in our society, the development of a novel pharmacologic intervention is a critically important public health goal. Recently, oleocanthal, a polyphenolic natural compound from extra virgin olive oil, has emerged as a potential therapeutic weapon for the treatment of inflammatory degenerative diseases. The goal of this study was to further evaluate the anti-inflammatory activity of oleocanthal in murine macrophages J774 and murine chondrocytes ATDC5 with a particular focus on the inhibition of gene expression of pro-inflammatory factors such as MIP-1α and IL-6. Main methodsATDC5 murine chondrogenic cells and murine macrophages J774 were used. J774 macrophages were tested with different doses of oleocanthal and cell viability was evaluated using the MTT assay. Western blot analysis was carried on in J774 cells using anti NOS2 antibody. Nitrite accumulation was determined in culture supernatant using the Griess reaction. MIP-1α and IL-6 mRNA levels were determined using SYBR Green-based quantitative RT-PCR. MIP-1α and IL-6 protein levels were evaluated using specific ELISA assay. Several cytokines, involved in the inflammatory response, were also tested by BioPlex assay. Key findingsFirst, oleocanthal inhibits LPS-induced NO production in J774 macrophages, without affecting cell viability. Moreover, it inhibits MIP-1α and IL-6 mRNA expression, as well as protein synthesis, in both ATDC5 chondrocytes and J774 macrophages. Oleocanthal also inhibits IL-1β, TNF-α and GM-CSF protein synthesis from LPS-stimulated macrophages. SignificanceOur data confirm a clear potent role of oleocanthal as anti-inflammatory therapeutic agent for future treatment of arthritis or other inflammatory diseases.

Fatty Acids Regulate Endothelial Lipase and Inflammatory Markers in Macrophages and in Mouse Aorta

Macrophage endothelial lipase (EL) is associated with increased atherosclerosis and inflammation. Because of their anti-inflammatory properties we hypothesized that n-3 fatty acids, in contrast to saturated fatty acids, would lower macrophages and arterial EL and inflammatory markers. Murine J774 and peritoneal macrophages were incubated with eicosapentaenoic acid or palmitic acid in the presence or absence of lipopolysaccaride (LPS). LPS increased EL mRNA and protein. Palmitic acid alone or with LPS dose-dependently increased EL mRNA and protein. In contrast, eicosapentaenoic acid dose-dependently abrogated effects of LPS or palmitic acid on increasing EL expression. EL expression closely linked to peroxisome proliferator activated receptor (PPAR)γ expression. Eicosapentaenoic acid blocked rosiglitazone (a PPARγ agonist)-mediated EL activation and GW9662 (a PPARγ antagonist)-blocked palmitic acid-mediated EL stimulation. Eicosapentaenoic acid alone or with LPS blunted LPS-mediated stimulation of macrophage proinflammatory interleukin-6, interleukin-12p40, and toll-like receptor-4 mRNA and increased anti-inflammatory interleukin-10 and mannose receptor mRNA. In vivo studies in low density lipoprotein receptor knockout mice showed that high saturated fat rich diets, but not n-3 diets, increased arterial EL, PPARγ, and proinflammatory cytokine mRNA. n-3 fatty acids, in contrast to saturated fatty acids, decrease EL in parallel with modulating pro- and anti-inflammatory markers, and these effects on EL link to PPARγ.

Development of an In Vitro Model for the Multi-Parametric Quantification of the Cellular Interactions between Candida Yeasts and Phagocytes

We developed a new in vitro model for a multi-parameter characterization of the time course interaction of Candida fungal cells with J774 murine macrophages and human neutrophils, based on the use of combined microscopy, fluorometry, flow cytometry and viability assays. Using fluorochromes specific to phagocytes and yeasts, we could accurately quantify various parameters simultaneously in a single infection experiment: at the individual cell level, we measured the association of phagocytes to fungal cells and phagocyte survival, and monitored in parallel the overall phagocytosis process by measuring the part of ingested fungal cells among the total fungal biomass that changed over time. Candida albicans, C. glabrata, and C. lusitaniae were used as a proof of concept: they exhibited species-specific differences in their association rate with phagocytes. The fungal biomass uptaken by the phagocytes differed significantly according to the Candida species. The measure of the survival of fungal and immune cells during the interaction showed that C. albicans was the more aggressive yeast in vitro, destroying the vast majority of the phagocytes within five hours. All three species of Candida were able to survive and to escape macrophage phagocytosis either by the intraphagocytic yeast-to-hyphae transition (C. albicans) and the fungal cell multiplication until phagocytes burst (C. glabrata, C. lusitaniae), or by the avoidance of phagocytosis (C. lusitaniae). We demonstrated that our model was sensitive enough to quantify small variations of the parameters of the interaction. The method has been conceived to be amenable to the high-throughput screening of mutants in order to unravel the molecular mechanisms involved in the interaction between yeasts and host phagocytes.