Macrophage-like THP-1 Cells Research Articles

2 Development of a sers-nanoparticle based system for the multiplex detection of vascular adhesion molecules

Surface-enhanced Raman spectroscopy (SERS) is an emerging molecular imaging technique suitable for the highly sensitive simultaneous detection of multiple analytes. We aimed to apply SERS for the multiplex detection of adhesion molecules in human umbilical vein endothelial cells and macrophages in vitro, as a step towards applying this technology for simultaneously detecting multiple biomarkers of vascular inflammation and atherosclerotic plaque instability in vivo. Immunofluorescent microscopy and flow cytometry identified the expression of intercellular adhesion molecule (ICAM)−1, vascular cell adhesion molecule (VCAM)−1 and p-Selectin on human umbilical vein endothelial cells (HUVECs) following stimulation with 10 ng/ml TNFα for 24 hours. Phorbol 12-myristate 13-acetate (PMA) differentiated THP-1 macrophage-like cells expressed only ICAM-1 in both unstimulated and 24 hours 10 ng/ml TNFα stimulated conditions. Gold nanoparticles were PEGylated and functionalized with Raman reporters, in order to endow a specific pre-determined SERS signal, alongside antibodies against the desired adhesion molecule targets. This provided ‘nanotags’ configured as follows (antibody/Raman reporter); anti-ICAM-1/BPE; anti-VCAM-1/PYOT, anti-p-Selectin/PPY, IgG Isotype Control/DP. TNFα stimulated HUVEC were exposed to a mixture of all nanotags simultaneously (final concentration of 20 ug/L) and were subsequently imaged using SERS microscopy. This rational allowed for the simultaneous detection and discrimination of ICAM-1, VCAM-1 and p-Selectin on activated HUVEC. Adhesion molecules were detectible as soon as 15 min following nanotag addition, while IgG control nanotags produced negligible signal at all concentrations and time points investigated. Following simultaneous exposure to all nanotags, SERS imaging identified the expression of ICAM-1 on PMA differentiated TNFα stimulated THP-1, with little-to-no contribution from anti-VCAM-1, anti-p-Selectin, or IgG control nanotags. Here we have developed a SERS-based molecular imaging methodology for the multiplex detection of adhesion molecules in vitro.

Dietary Buglossoides Arvensis Oil Increases Circulating n-3 Polyunsaturated Fatty Acids in a Dose-Dependent Manner and Enhances Lipopolysaccharide-Stimulated Whole Blood Interleukin-10-A Randomized Placebo-Controlled Trial.

Buglossoides arvensis (Ahiflower) oil is a dietary oil rich in stearidonic acid (20% SDA; 18:4 n-3). The present randomized, double blind, placebo-controlled clinical trial investigated the effects of three Ahiflower oil dosages on omega-3 polyunsaturated fatty acid (PUFA) content of plasma and mononuclear cells (MCs) and of the highest Ahiflower dosage on stimulated cytokine production in blood. Healthy subjects (n = 88) consumed 9.7 mL per day for 28 days of 100% high oleic sunflower oil (HOSO); 30% Ahiflower oil (Ahi) + 70% HOSO; 60% Ahi + 40% HOSO; and 100% Ahi. No clinically significant changes in blood and urine chemistries, blood lipid profiles, hepatic and renal function tests nor hematology were measured. Linear mixed models (repeated measures design) probed for differences in time, and time × treatment interactions. Amongst significant changes, plasma and MC eicosapentaenoic acid (EPA, 20:5 n-3) levels increased from baseline at day 28 in all Ahiflower groups (p < 0.05) and the increase was greater in all Ahiflower groups compared to the HOSO control (time × treatment interactions; p < 0.05). Similar results were obtained for α-linolenic acid (ALA, 18:3 n-3), eicosatetraenoic acid (ETA, 20:4 n-3), and docosapentaenoic acid (DPA, 22:5 n-3) content; but not docosahexaenoic acid (DHA, 22:6 n-3). Production of interleukin-10 (IL-10) was increased in the 100% Ahiflower oil group compared to 100% HOSO group (p < 0.05). IL-10 production was also increased in lipopolysaccharide (LPS)-stimulated M2-differentiated THP-1 macrophage-like cells in the presence of 20:4 n-3 or EPA (p < 0.05). Overall; this indicates that the consumption of Ahiflower oil is associated with an anti-inflammatory phenotype in healthy subjects.

Role of the LuxR family transcriptional regulator Lpg2524 in the survival of Legionella pneumophila in water.

The water-borne Gram-negative bacterium Legionella pneumophila (Lp) is the causative agent of Legionnaires' disease. Lp is typically transmitted to humans from water systems, where it grows inside amoebae. Survival of Lp in water is central to its transmission to humans. A transcriptomic study previously identified many genes induced by Lp in water. One such gene, lpg2524, encodes a putative LuxR family transcriptional regulator. It was hypothesized that this gene could be involved in the survival of Lp in water. Deletion of lpg2524 does not affect the growth of Lp in rich medium, in the amoeba Acanthamoeba castellanii, or in human macrophage-like THP-1 cells, showing that Lpg2524 is not required for growth in vitro and in vivo. Nevertheless, deletion of lpg2524 results in a faster colony-forming unit (CFU) reduction in an artificial freshwater medium, Fraquil, indicating that Lpg2524 is important for Lp to survive in water. Overexpression of Lpg2524 also results in a survival defect, suggesting that a precise level of this transcriptional regulator is essential for its function. However, our result shows that Lpg2524 is dispensable for survival in water when Lp is at a high cell density (109 CFU/mL), suggesting that its regulon is regulated by another regulator activated at high cell density.

Myosin II-Dependent Suppression of Podosomes by an ARNO-ARF1 Signaling Axis

Podosomes represent a special class of integrin-mediated cell-matrix adhesions formed by migrating and matrix degrading cells. Here, we demonstrated that in macrophage-like THP1 cells and in fibroblasts stimulated to produce podosomes, down-regulation of ARF1 by siRNA, and by pharmacological inhibitors led to striking podosome elimination. Treatments that induced podosome formation increased the level of GTP-bound ARF1. Furthermore, siRNA knockdown of the ARF1-GEF ARNO also resulted in dramatic podosome inhibition.

Evaluation of a human in vitro hepatocyte-NPC co-culture model for the prediction of idiosyncratic drug-induced liver injury: A pilot study.

Interactions between hepatocytes and immune cells as well as inflammatory episodes are frequently discussed to play a critical role in the alteration of the individual susceptibility to idiosyncratic drug-induced liver injury (iDILI). To evaluate this hypothesis and to face the urgent need for predictive in vitro models, we established two co-culture systems based on two human cell lines in presence or absence of pro-inflammatory factors (LPS, TNF), i.e. hepatoma HepG2 cells co-cultured with monocytic or macrophage-like THP-1 cells. HepG2 monocultures served as control scenario. Mono- or co-cultures were treated with iDILI reference substances (Troglitazone [TGZ], Trovafloxacin [TVX], Diclofenac [DcL], Ketoconazole [KC]) or their non-iDILI partner compounds (Rosiglitazone, Levofloxacin, Acetylsalicylic Acid, Fluconazole). The liver cell viability was subsequently determined via WST-Assay. An enhanced cytotoxicity (synergy) or a hormetic response compared to the drug effect in the HepG2 monoculture was considered as iDILI positive. TGZ synergized in co-cultures with monocytes without an additional pro-inflammatory stimulus, while DcL and KC showed a hormetic response. All iDILI drugs synergized with TNF in the simple HepG2 monoculture, indicating its relevance as an initiator of iDILI. KC showed a synergy when co-exposed to both, monocytes and LPS, while TVX and DcL showed a synergy under the same conditions with macrophages. All described iDILI responses were not observed with the corresponding non-iDILI partner compounds. Our first results confirm that an inflammatory environment increases the sensitivity of liver cells towards iDILI compounds and point to an involvement of pro-inflammatory factors, especially TNF, in the development of iDILI.

Podosome assembly is controlled by the GTPase ARF1 and its nucleotide exchange factor ARNO.

Podosomes represent a class of integrin-mediated cell-matrix adhesions formed by migrating and matrix-degrading cells. We demonstrate that in macrophage-like THP1 cells and fibroblasts stimulated to produce podosomes, down-regulation of the G-protein ARF1 or the ARF1 guanine nucleotide exchange factor, ARNO, by small, interfering RNA or pharmacological inhibitors led to striking podosome elimination. Concomitantly, treatments inducing podosome formation increased the level of guanosine triphosphate (GTP)-bound ARF1. ARNO was found to colocalize with the adhesive rings of podosomes, whereas ARF1 was localized to vesicular structures transiently contacting podosome rings. Inhibition of ARF1 led to an increase in RhoA-GTP levels and triggered assembly of myosin-IIA filaments in THP1 cells, whereas the suppression of myosin-IIA rescued podosome formation regardless of ARF1 inhibition. Finally, expression of constitutively active ARF1 in fibroblasts induced formation of putative podosome precursors: actin-rich puncta coinciding with matrix degradation sites and containing proteins of the podosome core but not of the adhesive ring. Thus, ARNO-ARF1 regulates formation of podosomes by inhibition of RhoA/myosin-II and promotion of actin core assembly.

Experimental Evolution of Mycobacterium tuberculosis in Human Macrophages Results in Low-Frequency Mutations Not Associated with Selective Advantage.

Isolates of the human pathogen Mycobacterium tuberculosis recovered from clinical samples exhibit genetic heterogeneity. Such variation may result from the stressful environment encountered by the pathogen inside the macrophage, which is the host cell tubercle bacilli parasitize. To study the evolution of the M. tuberculosis genome during growth inside macrophages, we developed a model of intracellular culture in which bacteria were serially passaged in macrophage-like THP-1 cells for about 80 bacterial generations. Genome sequencing of single bacterial colonies isolated before and after the infection cycles revealed that M. tuberculosis developed mutations at a rate of about 5.7 × 10−9 / bp/ generation, consistent with mutation rates calculated during in vivo infection. Analysis of mutant growth in macrophages and in mice showed that the mutations identified after the cyclic infection conferred no advantage to the mutants relative to wild-type. Furthermore, activity testing of the recombinant protein harboring one of these mutations showed that the presence of the mutation did not affect the enzymatic activity. The serial infection protocol developed in this work to study M. tuberculosis genome microevolution can be applied to exposure to stressors to determine their effect on genome remodeling during intra-macrophage growth.

Over-Expression of the Mycobacterial Trehalose-Phosphate Phosphatase OtsB2 Results in a Defect in Macrophage Phagocytosis Associated with Increased Mycobacterial-Macrophage Adhesion.

Trehalose-6-phosphate phosphatase (OtsB2) is involved in the OtsAB trehalose synthesis pathway to produce free trehalose and is strictly essential for mycobacterial growth. We wished to determine the effects of OtsB2 expression on mycobacterial phenotypes such as growth, phagocytosis and survival in macrophages. Mycobacterium bovis-bacillus calmette-guerin (BCG) over-expressing OtsB2 were able to better survive in stationary phase. Over-expression of OtsB2 led to a decrease in phagocytosis but not survival in THP-1 macrophage-like cells, and this was not due to a decrease in general macrophage phagocytic activity. Surprisingly, when we investigated macrophage–mycobacterial interactions by flow cytometry and atomic force microscopy, we discovered that BCG over-expressing OtsB2 have stronger binding to THP-1 cells than wild-type BCG. These results suggest that altering OtsB2 expression has implications for mycobacterial host–pathogen interactions. Macrophage–mycobacteria phagocytic interactions are complex and merit further study.

Vasodilator-Stimulated Phosphoprotein Activity Is Required for Coxiella burnetii Growth in Human Macrophages.

Coxiella burnetii is an intracellular bacterial pathogen that causes human Q fever, an acute flu-like illness that can progress to chronic endocarditis and liver and bone infections. Humans are typically infected by aerosol-mediated transmission, and C. burnetii initially targets alveolar macrophages wherein the pathogen replicates in a phagolysosome-like niche known as the parasitophorous vacuole (PV). C. burnetii manipulates host cAMP-dependent protein kinase (PKA) signaling to promote PV formation, cell survival, and bacterial replication. In this study, we identified the actin regulatory protein vasodilator-stimulated phosphoprotein (VASP) as a PKA substrate that is increasingly phosphorylated at S157 and S239 during C. burnetii infection. Avirulent and virulent C. burnetii triggered increased levels of phosphorylated VASP in macrophage-like THP-1 cells and primary human alveolar macrophages, and this event required the Cα subunit of PKA. VASP phosphorylation also required bacterial protein synthesis and secretion of effector proteins via a type IV secretion system, indicating the pathogen actively triggers prolonged VASP phosphorylation. Optimal PV formation and intracellular bacterial replication required VASP activity, as siRNA-mediated depletion of VASP reduced PV size and bacterial growth. Interestingly, ectopic expression of a phospho-mimetic VASP (S239E) mutant protein prevented optimal PV formation, whereas VASP (S157E) mutant expression had no effect. VASP (S239E) expression also prevented trafficking of bead-containing phagosomes to the PV, indicating proper VASP activity is critical for heterotypic fusion events that control PV expansion in macrophages. Finally, expression of dominant negative VASP (S157A) in C. burnetii-infected cells impaired PV formation, confirming importance of the protein for proper infection. This study provides the first evidence of VASP manipulation by an intravacuolar bacterial pathogen via activation of PKA in human macrophages.

Differences in Intracellular Fate of Two Spotted Fever Group Rickettsia in Macrophage-Like Cells.

Spotted fever group (SFG) rickettsiae are recognized as important agents of human tick-borne diseases worldwide, such as Mediterranean spotted fever (Rickettsia conorii) and Rocky Mountain spotted fever (Rickettsia rickettsii). Recent studies in several animal models have provided evidence of non-endothelial parasitism by pathogenic SFG Rickettsia species, suggesting that the interaction of rickettsiae with cells other than the endothelium may play an important role in pathogenesis of rickettsial diseases. These studies raise the hypothesis that the role of macrophages in rickettsial pathogenesis may have been underappreciated. Herein, we evaluated the ability of two SFG rickettsial species, R. conorii (a recognized human pathogen) and Rickettsia montanensis (a non-virulent member of SFG) to proliferate in THP-1 macrophage-like cells, or within non-phagocytic cell lines. Our results demonstrate that R. conorii was able to survive and proliferate in both phagocytic and epithelial cells in vitro. In contrast, R. montanensis was able to grow in non-phagocytic cells, but was drastically compromised in the ability to proliferate within both undifferentiated and PMA-differentiated THP-1 cells. Interestingly, association assays revealed that R. montanensis was defective in binding to THP-1-derived macrophages; however, the invasion of the bacteria that are able to adhere did not appear to be affected. We have also demonstrated that R. montanensis which entered into THP-1-derived macrophages were rapidly destroyed and partially co-localized with LAMP-2 and cathepsin D, two markers of lysosomal compartments. In contrast, R. conorii was present as intact bacteria and free in the cytoplasm in both cell types. These findings suggest that a phenotypic difference between a non-pathogenic and a pathogenic SFG member lies in their respective ability to proliferate in macrophage-like cells, and may provide an explanation as to why certain SFG rickettsial species are not associated with disease in mammals.

In vitro assessment of plutonium uptake and release using the human macrophage-like THP-1 cells

Plutonium (Pu) intake by inhalation is one of the major potential consequences following an accident in the nuclear industry or after improvised nuclear device explosion. Macrophages are essential players in retention and clearance of inhaled compounds. However, the extent to which these phagocytic cells are involved in these processes highly depends on the solubility properties of the Pu deposited in the lungs. Our objectives were to develop an in vitro model representative of the human pulmonary macrophage capacity to internalize and release Pu compounds in presence or not of the chelating drug diethylenetriaminepentaacetate (DTPA).The monocyte cell line THP-1 was used after differentiation into macrophage-like cells. We assessed the cellular uptake of various forms of Pu which differ in their solubility, as well as the release of the internalized Pu. Results obtained with differentiated THP-1 cells are in good agreement with data from rat alveolar macrophages and fit well with in vivo data. In both cell types, Pu uptake and release depend upon Pu solubility and in all cases DTPA increases Pu release.The proposed model may provide a good complement to in vivo animal experiments and could be used in a first assessment to predict the fraction of Pu that could be potentially trapped, as well as the fraction available to chelating drugs.

Long-chain monounsaturated fatty acid-rich fish oil attenuates the development of atherosclerosis in mouse models.

Fish oil-derived long-chain monounsaturated fatty acids (LCMUFA) containing chain lengths longer than 18 were previously shown to improve cardiovascular disease risk factors in mice. However, it is not known if LCMUFA also exerts anti-atherogenic effects. The main objective of the present study was to investigate the effect of LCMUFA on the development of atherosclerosis in mouse models. LDLR-KO mice were fed Western diet supplemented with 2% (w/w) of either LCMUFA concentrate, olive oil, or not (control) for 12 wk. LCMUFA, but not olive oil, significantly suppressed the development of atherosclerotic lesions and several plasma inflammatory cytokine levels, although there were no major differences in plasma lipids between the three groups. At higher doses 5% (w/w) LCMUFA supplementation was observed to reduce pro-atherogenic plasma lipoproteins and to also reduce atherosclerosis in ApoE-KO mice fed a Western diet. RNA sequencing and subsequent qPCR analyses revealed that LCMUFA upregulated PPAR signaling pathways in liver. In cell culture studies, apoB-depleted plasma from LDLR-K mice fed LCMUFA showed greater cholesterol efflux from macrophage-like THP-1 cells and ABCA1-overexpressing BHK cells. Our research showed for the first time that LCMUFA consumption protects against diet-induced atherosclerosis, possibly by upregulating the PPAR signaling pathway.

Glycosylation Changes Triggered by the Differentiation of Monocytic THP-1 Cell Line into Macrophages

The human acute monocytic leukemia cell line THP-1 is widely used as an in vitro phagocytic cell model because it exhibits several immune properties similar to native monocyte-derived macrophages. In this study, we investigated the alteration of N- and O-linked glycans as well as glycosphingolipids, during THP-1 differentiation, combining mass spectrometry, flow cytometry, and quantitative real-time PCR. Mass spectrometry revealed that macrophage differentiation led to a marked upregulation of expression of GM3 ganglioside as well as an increase in complex-type structures, particularly triantennary glycans, occurring at the expense of high-mannose N-glycans. Moreover, we observed a slight decrease in the proportion of multifucosylated N-glycans and α2,6-sialylation. The uncovered changes in glycosylation correlated with variations of gene expression of relevant glycosyltransferases and glycosidases including sialyltransferases, β-N-acetylglucosaminyltransferases, fucosyltransferases, and neuraminidase. Furthermore, using flow cytometry and antibodies directed against glycan structures, we confirmed that the alteration of glycosylation occurs at the cell surface of THP-1 macrophage-like cells. Altogether, we established that macrophagic maturation of THP-1 induces dramatic modifications of the surface glycosylation pattern that may result in differential interaction of monocytic and macrophagic THP-1 with immune or bacterial lectins.

Vermamoeba vermiformis-Aspergillus fumigatus relationships and comparison with other phagocytic cells

Free living amoebae (FLA) are protists ubiquitously present in the environment. Aspergillus fumigatus is a mould responsible for severe deep-seated infections, and that can be recovered in the same habitats as the FLA. By conducting coculture experiments and fungal incubation with amoebal supernatants, we report herein that Vermamoeba vermiformis, a FLA present in hospital water systems, promotes filamentation and growth of A. fumigatus. This finding is of particular importance to institutions whose water systems might harbor FLA and could potentially be used by immunocompromised patients. Also, the relationships between V. vermiformis and A. fumigatus were compared to those between this fungus and two other phagocytic cells: Acanthamoeba castellanii, another FLA, and macrophage-like THP-1 cells. After 4h of coincubation, the percentages of the three phagocytic cell types with adhered conidia were similar, even though the types of receptors between FLA and macrophagic cell seemed different. However, the percentage of THP-1 with internalized conidia was considerably lower (40%) in comparison with the two other cell types (100%). Thus, this study revealed that interactions between A. fumigatus and these three phagocytic cell types show similarities, even though it is premature to extrapolate these results to interpret relationships between A. fumigatus and macrophages.

A Single Dose of the Anti-Resorptive Peptide Human Calcitonin Paradoxically Augments Particle- and Endotoxin-Mediated Pro-Inflammatory Cytokine Production In Vitro.

The peptide hormone calcitonin (CT) is known to inhibit bone resorption and has previously been shown also to prevent particle-induced osteolysis, the leading cause of revision arthroplasty. In the present study, the influence of human CT on the initial inflammatory response to particulate wear debris or bacterial endotoxins, ultimately leading to osteoclast-mediated bone resorption, was analysed in human THP-1 macrophage-like cells. The cells were activated with either ultra-high molecular weight polyethylene (UHMWPE) particles or bacterial lipopolysaccharides (LPS) in order to simulate an osteolysis-associated inflammatory response. The cells were simultaneously treated with human CT (10(-9) M). Cytokine production of tumour necrosis factor (TNF)-α was quantified on both RNA and protein levels while interleukins (IL)-1β and IL-6 were measured as secreted protein only. Stimulation of the cells with either particles or LPS led to a dose- and time-dependent increase of TNF-α mRNA production and protein secretion of TNF-α, IL-1β, and IL-6. Application of CT mostly enhanced cytokine production as elicited by UHMWPE particles while a pronounced transient inhibitory effect on LPS-induced inflammation became evident at 24 h of incubation. Human CT displayed ambivalent effects on the wear- and LPS-induced production of pro-inflammatory cytokines. Thereby, the peptide primarily upregulated particle-induced inflammation while LPS-induced cytokine secretion was temporarily attenuated in a distinct manner. It needs to be evaluated whether the pro- or anti-inflammatory action of CT contributes to its known anti-resorptive effects. Thus, the therapeutic potential of the peptide in the treatment of either particle- or endotoxin-mediated bone resorption could be determined.

Examination of the effects of Campylobacter concisus zonula occludens toxin on intestinal epithelial cells and macrophages

BackgroundCampylobacter concisus is a Gram-negative bacterium that is associated with inflammatory bowel disease (IBD). Some C. concisus strains carry zonula occludens toxin (zot) gene which has polymorphisms. This study investigated the effects of C. concisus Zot on intestinal epithelial cells and macrophages using cell line models.MethodsCampylobacter concisuszot808T gene, a polymorphism that is associated with active IBD, was cloned and expressed in Escherichia coli. The effects of C. concisus Zot on intestinal epithelial barrier were examined using Caco-2 cell model. Apoptosis induced by C. concisus Zot in Caco-2 cells was assessed by measuring the levels of caspase 3/7. The production of pro-inflammatory cytokines induced by C. concisus Zot in HT-29 cells and in THP-1 macrophage-like cells was measured using ELISA kits. Whether exposure to C. concisus Zot can affect the responses of macrophages to E. coli K12 was also investigated.ResultsCampylobacter concisus Zot caused prolonged intestinal epithelial barrier damage, induced intestinal epithelial cell apoptosis, induced epithelial production of TNF-α and IL-8 and upregulated TNF-α in THP-1 macrophage-like cells. Pre-exposure to C. concisus Zot significantly enhanced the production of TNF-α and IL-8 as well as phagocytosis by THP-1 macrophage-like cells in response to E. coli K12.ConclusionThis study suggests that C. concisus Zot may have enteric pathogenic potential by damaging intestinal epithelial barrier, inducing intestinal epithelial and macrophage production of proinflammatory cytokines in particular TNF-α and enhancing the responses of macrophages to other enteric bacterial species.Electronic supplementary materialThe online version of this article (doi:10.1186/s13099-016-0101-9) contains supplementary material, which is available to authorized users.

The influence of calcitonin gene-related peptide on markers of bone metabolism in MG-63 osteoblast-like cells co-cultured with THP-1 macrophage-like cells under virtually osteolytic conditions.

BackgroundThe neuropeptide calcitonin gene-related peptide (CGRP) has been described to have an inhibitory effect on endotoxin- and wear particle-induced inflammation in the early stages of periprosthetic osteolysis. In the present study, the crosstalk between immune cells and osteoblasts in osteolytic conditions treated with CGRP has been analyzed to evaluate whether the anti-inflammatory properties of the peptide also have a beneficial, i.e. an anti-resorptive and osteo-anabolic impact on bone metabolism.MethodsMG-63 osteoblast-like cells were co-cultured with THP-1 macrophage-like cells stimulated with either ultra-high molecular weight polyethylene (UHMWPE) particles or different concentrations of bacterial lipopolysaccharides (LPS) and simultaneously treated with CGRP. Inflammation was monitored in terms of measuring the levels of tumor necrosis factor (TNF)-α secretion. Furthermore, the production of the osteoblast markers osteoprotegerin (OPG), receptor activator of nuclear factor κB ligand (RANKL), alkaline phosphatase (ALP) and osteopontin (OPN) was quantified. Also, ALP enzymatic activity was measured.ResultsStimulation of co-cultured THP-1 macrophages with either high levels of LPS or UHMWPE induced the secretion of TNF-α which could be inhibited by CGRP to a great extent. However, no remarkable changes in the OPG/RANKL ratio or bone ALP activity were observed. Interestingly, OPN was exclusively produced by THP-1 cells, thus acting as a marker of inflammation. In addition, TNF-α production in THP-1 single cell cultures was found to be considerably higher than in co-cultured cells.ConclusionsIn the co-culture system used in the present study, no obvious relation between inflammation, its mitigation by CGRP, and the modulation of bone metabolism became evident. Nonetheless, the results suggest that during the onset of periprosthetic osteolysis the focus might lie on the modulation of inflammatory reactions. Possibly, implant-related inflammation might merely have an impact on osteoclast differentiation rather than on the regulation of osteoblast activity.Electronic supplementary materialThe online version of this article (doi:10.1186/s12891-016-1044-5) contains supplementary material, which is available to authorized users.

Abstract 266: Role of Marine Long-chain Monounsaturated Fatty Acid in Cardiovascular Disease Prevention and Possible Mechanism to Reduce the Atherosclerosis

Numerous studies have shown cardiovascular benefits of fish oil, and most of these favorable effects have been attributed to omega-3 fatty acids. Fish oils, however, also contain varying amounts of other unusual types of fatty acids, such as long-chain monounsaturated fatty acids (LCMUFA; i.e., C20:1 and C22:1 isomers combined). Compared with well-studied omega-3, limited information is available on the role of LCMUFA on cardiovascular disease. In the current study, we examined the effect of saury fish oil-derived LCMUFA concentrate on the pathogenesis of atherosclerosis in ApoE-deficient and LDLR-deficient mice. Male ApoE-deficient mice were fed a western diet supplemented with 5% LCMUFA concentrate or not (control) for 12 weeks. En face Sudan IV staining of the aorta revealed that LCMUFA significantly suppressed the development of atherosclerotic lesions, and down-regulated inflammatory gene expression. In LDLR-deficient female mice, we also observed that 12-week supplementation of 2% LCMUFA on a western diet significantly decreased atherosclerosis lesion areas and accumulation of macrophages, compared with western diet (control) or western diet supplemented with 2% olive oil enriched in shorter-chain MUFA oleic acid (C18:1). There were no differences, however, in plasma or hepatic lipid profiles between the three groups. LCMUFA, but not olive oil, also significantly suppressed several plasma inflammatory cytokine levels. RNA sequencing and subsequent qPCR analyses revealed that LCMUFA upregulated PPAR signaling pathways in liver. In cell culture studies, apoB-depleted plasma from the LCMUFA group promoted cholesterol efflux from macrophage-like THP-1 cells and ABCA1-overexpressing BHK cells. LCMUFA also suppressed LPS-induced inflammatory cytokine production by macrophages. In conclusion, our research showed for the first time that LCMUFA consumption could protects against atherosclerosis, possibly by upregulating the PPAR signaling pathway. Omega-3 fatty acids are generally considered the major active components in fish lipids, but our findings provide new insights into potential health effect of LCMUFA-rich fish oil and suggest that LCMUFA-rich fish oil may also be beneficial for reducing cardiovascular disease risk.

Kinetic Measures of Macrophage Chemotaxis

Abstract Macrophages are crucial regulators of homeostasis, however, their functions can be compromised, leading to a wide variety of diseases such as diabetes, fibrosis, atherosclerosis and tumor metastasis. Macrophage recruitment by cytokines is an essential step of their function. The main obstacles for studying macrophage chemotaxis in vitro include low resident tissue macrophage cell number, lengthy isolation periods, and low percentage of migrating cells. In this report we illustrate a novel, fully automated, kinetic, label-free method for measuring macrophage chemotaxis. This method requires significantly fewer cells compared to traditional methods, thus allowing for increased throughput. Using the human monocyte cell line, THP-1, we first examined the directed migration of untreated vs. PMA-differentiated (macrophage-like) THP-1 cells. Using as few as 5000 cells per well, we observed a significantly different chemotactic response depending on treatment: pre-activated THP-1 had minimal to no response to C5a while differentiated THP-1 had a strong concentration-dependent response. Primary human PBMC-derived Macrophages were also evaluated. Using 2000 cells per well we found that both M1 (pro-inflammatory) and M2 (pro-regenerative) macrophages consistently responded to C5a with minimal, yet significant responses towards Rantes, SDF-1α, CXCL10, MDC and M-CSF. M1 cells exhibited slower response compared to M2 cells. Our data also revealed a significant alteration in kinetic responses depending on C5a concentration, with higher concentrations of C5a eliciting a slower response than lower concentrations, thus illustrating the requirement for kinetic measurements when investigating macrophage chemotaxis.

Species diversity and cytotoxic potency of airborne sterigmatocystin-producing Aspergilli from the section Versicolores

This study presents the distribution and species diversity of sterigmatocystin-producing Aspergilli from the section Versicolores in the indoor air of apartment-AP, basements-BS and grain mill-GM in Croatia, as well as the cytotoxic potency of isolates. The species comprised 0.7–20% of total airborne fungi detected in the AP, 11–55% in the BS, and 0–2% in the GM. Based on CaM sequences, seven species were identified; dominant were Aspergillus jensenii and Aspergillus creber, followed by Aspergillus protuberus, Aspergillus venenatus, Aspergillus tennesseensis, Aspergillus amoenus, Aspergillus griseoaurantiacus and three undescribed species. All of the identified species produced sterigmatocystin-STC (HPLC/UV-VIS); A. griseoaurantiacus (208.29μg/mL) and A. jensenii (1.192–133.63μg/mL) produced the highest levels, the lowest were detected in A. protuberus and A. tennesseensis (0.117–2.749μg/mL). Lower species diversity was obtained in the GM due to overgrowth with more propulsive fungi. Relatively high STC levels (0.06–2.35μg/g) detected in 52% of GM dust samples confirmed the presence of STC-producers, although this STC cannot be exclusively attributed to Aspergilli (Versicolores). STC and the majority of STC-producing Aspergilli were cytotoxic to human lung A549 cells (IC50 0.9–2.3μg/mL) and THP-1 macrophage-like cells (IC50 0.3–0.6μg/mL) in relatively low concentrations suggesting that humans can be at high risk during chronic exposure.