Macrophage-differentiated THP-1 Cells Research Articles

Effects of Fasting on THP1 Macrophage Metabolism and Inflammatory Profile.

Fasting can affect the body's inflammatory response, and this has been linked to potential health benefits, including improvements for people with rheumatic diseases. In this work, we evaluated, in vitro, how changes in nutrient availability alter the inflammatory response of macrophages. Macrophage-differentiated THP1 cells were cultured, deprived of FCS or subjected to cycles of FCS deprivation and restoration to mimic intermittent fasting. Changes in the macrophage phenotype, the cells' response to inflammatory stimuli and the level of mitochondrial alteration were assessed. The results indicate that while periods of serum starvation are associated with a decrease in IL1β and TNFα expression, consistent with an anti-inflammatory response, intermittent serum starvation cycles promote a pro-inflammatory phenotype. Rapid changes in reducing capacity and mitochondrial response were also observed. Of note, while some changes, such as the production of oxygen free radicals, were reversed with refeeding, others, such as a decrease in reducing capacity, were maintained and even increased. This study shows that different fasting protocols can have diverging effects and highlights that time-limited nutrient changes can significantly affect macrophage functions in cell cultures. These findings help elucidate some of the mechanisms by which specific fasting dietary interventions could help control inflammatory diseases.

Abstract 5365: Characterizing signaling in macrophage-GBM cell coculture

Abstract Macrophages are a type of immune cell that play a crucial role in the tumor microenvironment. In tumors, macrophages can be polarized towards a pro-inflammatory phenotype, referred to as M1, or towards an anti-inflammatory and tumor-promoting phenotype, referred to as M2. M2 macrophages promote tumor growth by secreting cytokines that increase tumor proliferation, angiogenesis, and dampen the immune response from other immune cells such as T cells and dendritic cells. The balance between M1 and M2 macrophages in the tumor microenvironment is critical in determining the outcome of cancer progression and response to therapy. By understanding the pathways that lead to macrophage polarization, we can develop therapeutics that inhibit this process and support an anti-tumor macrophage response. Co-culture is a powerful model to study interactions between different cell types. We created co-cultures with macrophages and glioblastoma multiforme (GBM) cells to understand how these cell-cell interactions can lead to altered cell phenotype. Several studies have looked at the interaction of two populations using a transwell system where one cell population is placed in a well with a permeable membrane and the other population is placed below, allowing for the exchange of soluble factors. However, this does not account for an important mode of cell-cell interaction, which is physical contact. Therefore, we tested how macrophages derived from primary monocytes polarized in the context of conditioned media (supernatant), cell lysate, and direct co-culture. We used U87-MG as a model GBM cell line. We assayed the M2 phenotype by using flow cytometry with CD163 as a marker. Our findings show that direct co-culture is crucial for promoting macrophage M2 polarization. Using our in vitro model for tumor-induced macrophage polarization, we conducted phosphoproteomic analysis to characterize signals associated with this phenotypic change. To study how co-culture induced differential signaling in tumor populations and macrophages, we devised a method to fix cells to preserve cell signaling, followed by separation using flow cytometry and LC-MS/MS analysis of individual cell populations. We created co-cultures of U87-MG tumor cells and macrophage-differentiated THP-1 cells, a monocyte cell line. We then tested whether our method could reliably separate the different populations, whether we could detect altered signals due to co-culture, and showcase how separating cell populations yields information that could not be gathered from bulk analysis of co-cultures. Our data shows that the analysis was highly reproducible and that the signaling from cells separated from co-culture was highly similar to signaling in cells that were grown individually. However, some phosphorylation residues were uniquely upregulated in the co-culture condition, pointing to signaling that may occur in response to tumor interaction. Citation Format: Alicia D. D'Souza, Rachit Mukkamala, Ryuhjin Ahn, Forest M. White. Characterizing signaling in macrophage-GBM cell coculture [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5365.

The lncRNA H19/miR-29a-3p/SNIP1/c-myc regulatory axis is involved in pulmonary fibrosis induced by Nd2O3.

Some rare earth elements are occupational and environmental toxicants and can cause organ and systemic damage; therefore, they have attracted global attention. Neodymium oxide (Nd2O3) is a rare earth element that is refined and significantly utilized in China. The long noncoding RNA (lncRNA) H19 is encoded by the H19/IGF2 imprinted gene cluster located on human chromosome 11p15.5. H19 has become a research focus due to its ectopic expression leading to the promotion of fibrosis. However, the mechanisms by which it causes pulmonary fibrosis are elusive. This investigation indicates that biologically active Nd2O3 increases H19, SNIP1, and c-myc, decreases miR-29a-3p, accelerates macrophage M2 polarization, and causes pulmonary fibrosis in mice lung tissues. In macrophage-differentiated THP-1 cells, Nd2O3 (25 μg/ml) enhanced H19, SNIP1, and c-myc, reduced miR-29a-3p, accelerated macrophages M2 polarization, and stimulated fibrogenic cytokine (TGF-β1) secretion. Furthermore, the coculturing of Nd2O3-treated macrophage-differentiated THP-1 cells. And human embryonic lung fibroblast cells activated lung fibroblast, which increases the levels of collagen I, α-SMA, p-Smad2/3, and Smad4, whereas H19 knockdown or miR-29a-3p upregulation in macrophages had opposite effects. Moreover, it was revealed that H19/miR-29a-3p/SNIP1/c-myc regulatory axis is involved in pulmonary fibrosis induced by Nd2O3. Therefore, this study provides new molecular insights into the mechanism of pulmonary fibrosis by Nd2O3.

Group A Streptococcus cation diffusion facilitator proteins contribute to immune evasion by regulating intracellular metal concentrations

Cation diffusion facilitators (CDFs) are a large family of divalent metal transporters with broad specificities that contribute to intracellular metal homeostasis and toxicity in bacterial pathogens. Streptococcus pyogenes (Group A Streptococcus [GAS]) expresses two homologous CDF efflux transporters, MntE and CzcD, which selectively transport Mn and Zn, respectively. We discovered that the MntE- and CzcD-deficient strains exhibited a marked decrease in the viability of macrophage-differentiated THP-1 cells and neutrophils. In addition, the viability of mice infected with both deficient strains markedly increased. Consistent with a previous study, our results suggest that MntE regulates the PerR-dependent oxidative stress response by maintaining intracellular Mn levels and contributing to the growth of GAS. The maturation and proteolytic activity of streptococcal cysteine protease (SpeB), an important virulence factor in GAS, has been reported to be abrogated by zinc and copper. Zn inhibited the maturation and proteolytic activity of SpeB in the culture supernatant of the CzcD-deficient strain. Furthermore, Mn inhibited SpeB maturation and proteolytic activity in a MntE-deficient strain. Since the host pathogenicity of the SpeB-deficient strain was significantly reduced, maintenance of intracellular manganese and zinc levels in the GAS via MntE and CzcD may not only confer metal resistance to the bacterium, but may also play an essential role in its virulence. These findings provide new insights into the molecular mechanisms of pathogenicity, which allow pathogens to survive under stressful conditions associated with elevated metal ion concentrations during host infection.

Micronized Palmitoylethanolamide, Hempseed Oil, and Maritime Pine Bark Dry Extract (Pelvipea®) for Pelvic Pain: An In Vitro Study for Urothelial Inflammation Treatment.

Urothelial inflammation plays a key role in the pathogenesis of chronic pelvic pain due to its origin in the bladder. The aim of this study was to evaluate the efficacy of a patent-pending formulation (Pelvipea®) composed of micronized palmitoylethanolamide (PEA), hempseed oil, and maritime pine bark dry extract in reducing urothelial inflammation, as well as the effect of each ingredient individually, in order to define the synergistic effect of the three ingredients. An in vitro bladder urothelium model composed of the T24 cell line was exposed to a conditioned media obtained by treating macrophage-differentiated THP-1 cells with different concentrations of the functional ingredients and a mixture of them in the presence of the pro-inflammatory stimulus of Escherichia coli. Cells exposed only to the inflammatory stimulus in the absence of pre-treatment were considered as a positive control for inflammation. The impact of each functional ingredient and their mixture on inflammation was evaluated by the determination of transcription factor NF-kB and of pro-inflammatory cytokine expression. Statistical analysis was performed using the t-test, comparing the mixture and the single ingredients for every condition tested. All results were reported as fold change (mean ± standard deviation), the ratio between the values obtained from the respective treatments for inflammation control. The three functional ingredients did not induce negative effects on THP-1 cell vitality. The levels of NF-kB were reduced following treatment with hempseed oil, maritime pine bark dry extract, and the mixture at all tested concentrations, and with micronized PEA from 25 to 200 μg/mL. Treatment with the mixture resulted in the lowest expression levels of interleukins (IL)-1β, IL-6, and IL-8 compared to the single functional ingredients at a concentration of 230 μg/mL, with values of 0.08 (±0.00), 0.01 (±0.00), and 0.32 (±0.01), respectively. The mixture of micronized PEA, hempseed oil, and maritime pine bark dry extract (Pelvipea®) at 230 μg/mL showed the best efficacy in urothelial IL-1β, IL-6, and IL-8 reduction compared with the singular components. This formulation may represent a promising therapeutic option to relieve painful symptoms originating in the bladder. However, in vivo studies are needed to confirm these results.

Nanoencapsulation of Gla-Rich Protein (GRP) as a Novel Approach to Target Inflammation.

Chronic inflammation is a major driver of chronic inflammatory diseases (CIDs), with a tremendous impact worldwide. Besides its function as a pathological calcification inhibitor, vitamin K-dependent protein Gla-rich protein (GRP) was shown to act as an anti-inflammatory agent independently of its gamma-carboxylation status. Although GRP’s therapeutic potential has been highlighted, its low solubility at physiological pH still constitutes a major challenge for its biomedical application. In this work, we produced fluorescein-labeled chitosan-tripolyphosphate nanoparticles containing non-carboxylated GRP (ucGRP) (FCNG) via ionotropic gelation, increasing its bioavailability, stability, and anti-inflammatory potential. The results indicate the nanosized nature of FCNG with PDI and a zeta potential suitable for biomedical applications. FCNG’s anti-inflammatory activity was studied in macrophage-differentiated THP1 cells, and in primary vascular smooth muscle cells and chondrocytes, inflamed with LPS, TNFα and IL-1β, respectively. In all these in vitro human cell systems, FCNG treatments resulted in increased intra and extracellular GRP levels, and decreased pro-inflammatory responses of target cells, by decreasing pro-inflammatory cytokines and inflammation mediators. These results suggest the retained anti-inflammatory bioactivity of ucGRP in FCNG, strengthening the potential use of ucGRP as an anti-inflammatory agent with a wide spectrum of application, and opening up perspectives for its therapeutic application in CIDs.

Multi-walled carbon nanotubes trigger lysosome-dependent cell death (pyroptosis) in macrophages but not in neutrophils

Carbon nanotubes (CNTs) have been extensively investigated, and several studies have shown that multi-walled CNTs can trigger inflammation and fibrosis in animal models. However, while neutrophils are involved in inflammation, most in vitro studies have addressed macrophages. Here we explored the impact of three MWCNTs with varying morphology (i.e. long and rigid versus short and/or tangled) on primary human macrophages and macrophage-differentiated THP-1 cells versus primary human neutrophils and neutrophil-differentiated HL-60 cells. We found that long and rigid MWCNTs triggered caspase-dependent cell death in macrophages, accompanied by NLRP3 inflammasome activation and gasdermin D (GSDMD)-mediated release of pro-inflammatory IL-1β. The release of IL-1β was suppressed by disulfiram, an FDA-approved drug known to act as an inhibitor of membrane pore formation by GSDMD. Evidence of autophagic cell death was noted in macrophages exposed to higher concentrations of the long and rigid MWCNTs. Furthermore, lysosomal damage with cytosolic release of cathepsin B was observed in macrophages exposed to the latter MWCNTs. On the other hand, there was little evidence of uptake of MWCNTs in neutrophils and the cells failed to undergo MWCNT-triggered cell death. Our studies have demonstrated that long and rigid MWCNTs trigger pyroptosis in human macrophages.

Proinflammatory response caused by lead nanoparticles triggered by engulfed nanoparticles.

In this study, the cellular effects of lead (Pb) nanoparticles with a primary particle size of 80 nm were evaluated in two types of cell lines: human lung carcinoma A549 and macrophage-differentiated THP-1 cells (dTHP-1). The cellular responses induced by the Pb nanoparticles varied among the cell types. Exposure to Pb nanoparticles for 24 h at a concentration of 100 μg/ml induced interleukin-8 (IL-8) expression in dTHP-1 cells. Induction of IL-8 expression in A549 was lower than dTHP-1 cells. Pb nanoparticles also induced the gene expression of heme oxygenase-1 in dTHP-1 cells but not in A549 cells. Though cellular uptake of Pb nanoparticles was observed in both the cell types, the amount of internalized Pb particles was lower in A549 cells than that in dTHP-1 cells. Gene expression of metallothionein 2A was remarkably enhanced by Pb nanoparticle exposure in dTHP-1 cells. Compared with Pb nanoparticles, induction of cytokines caused by lead nitrate (Pb[NO3 ]2 ), a water-soluble Pb compound, was smaller. In conclusion, the present study revealed that Pb nanoparticles induced a stronger cellular response than Pb(NO3 )2 , primarily by eliciting cytokine production, in a cell type-dependent manner.

Carrageenan from red algae: an application in the development of inhalable tuberculosis therapy targeting the macrophages.

Macrophages have unique surface receptors that might recognize preferentially several moieties present on the surface of infecting organisms, including in the bacterial cell wall. Benefiting from a similar composition regarding the referred moieties, polysaccharides might be good candidates to compose the matrix of drug carriers aimed at macrophage targeting, as they can use the same recognition pathways of the infecting organisms. Carrageenan (CRG), a polysaccharide extracted from red edible seaweed, is an interesting possibility for the approach of directly targeting alveolar macrophages, as its composition is reported to be recognized by several macrophage lectin receptors. Inhalable starch/CRG microparticles were successfully produced, effectively associating isoniazid (96%) and rifabutin (74%) simultaneously. Furthermore, the produced microparticles presented adequate aerodynamic properties for pulmonary delivery with potential to reach the respiratory zone, with a mass median aerodynamic diameter (MMAD) between 3.3 and 3.9μm. It was further demonstrated that the antitubercular activity of the drugs remained unchanged after encapsulation. The formulation evidenced no cytotoxic effects on lung epithelial cells (A549), although mild toxicity was observed on macrophage-differentiated THP-1 cells for the drug-loaded formulation. Starch/CRG microparticles also exhibited a propensity to be captured by macrophages in a dose-dependent manner, as well as an ability to activate the target cells. This work provides indications on the potential of the starch/CRG carriers to interact with macrophages, thus providing a platform for drug delivery in the context of macrophage intracellular diseases. Additionally, if tuberculosis is focused, these microparticles can be used as inhalable drug carriers. Graphical abstractz.

Role of Shiga Toxins in Cytotoxicity and Immunomodulatory Effects of Escherichia coli O157:H7 during Host-Bacterial Interactions in vitro.

Enterohemorrhagic Escherichia coli (EHEC) strains are food-borne pathogens that can cause different clinical conditions. Shiga toxin 2a and/or 2c (Stx2)-producing E. coli O157:H7 is the serotype most frequently associated with severe human disease. In this work we analyzed the hypothesis that host cells participate in Stx2 production, cell damage, and inflammation during EHEC infection. With this aim, macrophage-differentiated THP-1 cells and the intestinal epithelial cell line HCT-8 were incubated with E. coli O157:H7. A time course analysis of cellular and bacterial survival, Stx2 production, stx2 transcription, and cytokine secretion were analyzed in both human cell lines. We demonstrated that macrophages are able to internalize and kill EHEC. Simultaneously, Stx2 produced by internalized bacteria played a major role in macrophage death. In contrast, HCT-8 cells were completely resistant to EHEC infection. Besides, macrophages and HCT-8 infected cells produce IL-1β and IL-8 inflammatory cytokines, respectively. At the same time, bacterial stx2-specific transcripts were detected only in macrophages after EHEC infection. The interplay between bacteria and host cells led to Stx production, triggering of inflammatory response and cell damage, all of which could contribute to a severe outcome after EHEC infections.

Inhalable chitosan microparticles for simultaneous delivery of isoniazid and rifabutin in lung tuberculosis treatment

The direct delivery of antibiotics to the lung has been considered an effective approach to treat pulmonary tuberculosis, which represents approximately 80% of total cases. In this sense, this work aimed at producing inhalable chitosan microparticles simultaneously associating isoniazid and rifabutin, for an application in pulmonary tuberculosis therapy. Spray-dried chitosan microparticles were obtained with adequate flow properties for deep lung delivery (aerodynamic diameter of 4 µm) and high drug association efficiencies (93% for isoniazid and 99% for rifabutin). The highest concentration of microparticles that was tested (1 mg/mL) decreased the viability of macrophage-differentiated THP-1 cells to around 60% after 24 h exposure, although no deleterious effect was observed in human alveolar epithelial (A549) cells. The release of LDH was, however, increased in both cells. Chitosan microparticles further evidenced capacity to activate macrophage-like cells, inducing cytokine secretion well above basal levels. Moreover, the propensity of macrophages to internalize microparticles was demonstrated, with uptake levels over 90%. Chitosan microparticles also inhibited bacterial growth by 96%, demonstrating that the microencapsulation preserved drug antibacterial activity in vitro. Overall, the obtained data suggest the potential of chitosan microparticles for inhalable lung tuberculosis therapy.

Macrophage in vitro Response on Hybrid Coatings Obtained by Matrix Assisted Pulsed Laser Evaporation

The improvement in the research area of the implant by surface functionalization when correlated with the biological response is of major interest in the biomedical field. Based on the fact that the inflammatory response is directly involved in the ultimate response of the implant within the body, it is essential to study the macrophage-material interactions. Within this context, we have investigated the composite material-macrophage cell interactions and the inflammatory response to these composites with amorphous hydroxyapatite (HA), Lactoferrin (Lf), and polyethylene glycol-polycaprolactone (PEG-PCL) copolymer. All materials are obtained by Matrix Assisted Pulsed Laser Evaporation (MAPLE) technique and characterized by Atomic Force Microscopy and Scanning Electron Microscopy. Macrophage-differentiated THP-1 cells proliferation and metabolic activity were assessed by qualitative and quantitative methods. The secretion of tumor necrosis factor alpha (TNF-α) and interleukin 10 (IL-10) cytokine, in the presence and absence of the inflammatory stimuli (bacterial endotoxin; lipopolysaccharide (LPS)), was measured using an ELISA assay. Our results revealed that the cellular response depended on the physical-chemical characteristics of the coatings. Copolymer-HA-Lf coatings led to low level of pro-inflammatory TNF-α, the increased level of anti-inflammatory IL-10, and the polarization of THP-1 cells towards an M2 pro-reparative phenotype in the presence of LPS. These findings could have important potential for the development of composite coatings in implant applications.

Inhalable Fucoidan Microparticles Combining Two Antitubercular Drugs with Potential Application in Pulmonary Tuberculosis Therapy.

The pulmonary delivery of antitubercular drugs is a promising approach to treat lung tuberculosis. This strategy not only allows targeting the infected organ instantly, it can also reduce the systemic adverse effects of the antibiotics. In light of that, this work aimed at producing fucoidan-based inhalable microparticles that are able to associate a combination of two first-line antitubercular drugs in a single formulation. Fucoidan is a polysaccharide composed of chemical units that have been reported to be specifically recognised by alveolar macrophages (the hosts of Mycobacterium). Inhalable fucoidan microparticles were successfully produced, effectively associating isoniazid (97%) and rifabutin (95%) simultaneously. Furthermore, the produced microparticles presented adequate aerodynamic properties for pulmonary delivery with potential to reach the respiratory zone, with a mass median aerodynamic diameter (MMAD) between 3.6–3.9 µm. The formulation evidenced no cytotoxic effects on lung epithelial cells (A549), although mild toxicity was observed on macrophage-differentiated THP-1 cells at the highest tested concentration (1 mg/mL). Fucoidan microparticles also exhibited a propensity to be captured by macrophages in a dose-dependent manner, as well as an ability to activate the target cells. Furthermore, drug-loaded microparticles effectively inhibited mycobacterial growth in vitro. Thus, the produced fucoidan microparticles are considered to hold potential as pulmonary delivery systems for the treatment of tuberculosis.

Specific Interactions Measured by AFM on Living Cells between Peroxiredoxin-5 and TLR4: Relevance for Mechanisms of Innate Immunity

Inflammation is a pathophysiological response of innate immunity to infection or tissue damage. This response is among others triggered by factors released by damaged or dying cells, termed damage-associated molecular pattern (DAMP) molecules that act as danger signals. DAMPs interact with pattern recognition receptors (PRRs) to contribute to the induction of inflammation. However, how released peroxiredoxins (PRDXs) are able to activate PRRs, such as Toll-like receptors (TLRs), remains elusive. Here, we used force-distance curve-based atomic force microscopy to investigate the molecular mechanisms by which extracellular human PRDX5 can activate a proinflammatory response. Single-molecule experiments demonstrated that PRDX5 binds to purified TLR4 receptors, on macrophage-differentiated THP-1 cells, and on human TLR4-transfected CHO cells. These findings suggest that extracellular PRDX5 can specifically trigger a proinflammatory response. Moreover, our work also revealed that PRDX5 binding induces a cellular mechanoresponse. Collectively, this study provides insights into the role of extracellular PRDX5 in innate immunity.

Functional Analyses of the Crohn's Disease Risk Gene LACC1.

BackgroundGenetic variation in the Laccase (multicopper oxidoreductase) domain-containing 1 (LACC1) gene has been shown to affect the risk of Crohn’s disease, leprosy and, more recently, ulcerative colitis and juvenile idiopathic arthritis. LACC1 function appears to promote fatty-acid oxidation, with concomitant inflammasome activation, reactive oxygen species production, and anti-bacterial responses in macrophages. We sought to contribute to elucidating LACC1 biological function by extensive characterization of its expression in human tissues and cells, and through preliminary analyses of the regulatory mechanisms driving such expression.MethodsWe implemented Western blot, quantitative real-time PCR, immunofluorescence microscopy, and flow cytometry analyses to investigate fatty acid metabolism-immune nexus (FAMIN; the LACC1 encoded protein) expression in subcellular compartments, cell lines and relevant human tissues. Gene-set enrichment analyses were performed to initially investigate modulatory mechanisms of LACC1 expression. A small-interference RNA knockdown in vitro model system was used to study the effect of FAMIN depletion on peroxisome function.ResultsFAMIN expression was detected in macrophage-differentiated THP-1 cells and several human tissues, being highest in neutrophils, monocytes/macrophages, myeloid and plasmacytoid dendritic cells among peripheral blood cells. Subcellular co-localization was exclusively confined to peroxisomes, with some additional positivity for organelle endomembrane structures. LACC1 co-expression signatures were enriched for genes involved in peroxisome proliferator-activated receptors (PPAR) signaling pathways, and PPAR ligands downregulated FAMIN expression in in vitro model systems.ConclusionFAMIN is a peroxisome-associated protein with primary role(s) in macrophages and other immune cells, where its metabolic functions may be modulated by PPAR signaling events. However, the precise molecular mechanisms through which FAMIN exerts its biological effects in immune cells remain to be elucidated.

Phagocyte interactions with Mycobacterium tuberculosis — Simultaneous analysis of phagocytosis, phagosome maturation and intracellular replication by imaging flow cytometry

Utilization of compounds that enhance the innate immune response against Mycobacterium tuberculosis is an attractive strategy for combating tuberculosis in the post-antibiotic era. Thus, it is crucial to develop methods that can be used to screen for such compounds and to investigate their mechanisms of action. Here, we used imaging flow cytometry (ImageStreamX Mk II), which enables rapid quantification of microscopic images in flow, to study the interaction between phagocytes and M. tuberculosis. Macrophage-differentiated THP-1 cells were infected with GFP-expressing M. tuberculosis H37Ra, and methods for rapidly assessing phagocytosis, phagosome maturation, and bacterial replication inside the cells were developed and evaluated. These aspects of innate immunity are essential in determining the outcome of mycobacterial infection of phagocytes. The technique was found effective for monitoring phagocytosis of mycobacteria, phagosomal acidification and phagolysosomal fusion, as well as for measuring mycobacterial replication inside the cells. Several of these aspects could be analyzed simultaneously in the same sample, providing a great deal of information about the phagocyte–mycobacterial interaction at once. Thus, this method has great potential to be useful both for basic research questions and for evaluating compounds that enhance the innate immune response against M. tuberculosis.

Stimulation of macrophages with the β-glucan produced by aureobasidium pullulans promotes the secretion of tumor necrosis factor-related apoptosis inducing ligand (TRAIL).

A β-glucan produced by Aureobasidium pullulans (AP-PG) is consisting of a β-(1,3)-linked main chain with β-(1,6)-linked glucose side residues. Various β-glucans consisting of β-(1,3)-linked main chain including AP-PG are believed to exhibit anti-tumor activities, and actually, anti-tumor activities of AP-PG in mice have been demonstrated. In this study, we demonstrate that stimulation with AP-PG induces TRAIL expression in mouse and human macrophage-like cell lines. TRAIL is known to be a cytokine which specifically induces apoptosis in transformed cells, but not in untransformed cells. The expression of TRAIL mRNA after stimulation with AP-PG was increased in RAW264.7 cells, Mono Mac 6 cells, and macrophage-differentiated THP-1 cells. The mRNA expression of TNF-α and FasL is only weakly increased after stimulation with AP-PG. The induction activity of TRAIL by curdlan, a bacterial β-glucan, was very similar to that by AP-PG in RAW264.7 cells, but weaker in macrophage-differentiated THP-1 cells. Activation of caspases was found in HeLa cells after treatment with the supernatant of cultured medium from AP-PG-stimulated Mono Mac 6 cells, and was inhibited by the anti-TRAIL neutralizing antibody. These findings suggest that the stimulation with AP-PG effectively induces TRAIL in macrophages, and that it may be related to apoptosis induction of tumor cells.

Exposure to an organometal compound stimulates adipokine and cytokine expression in white adipose tissue

Objective White adipose tissue (WAT) is now considered a defined tissue capable of interactions with other organ systems. WAT role in elevating the level of systemic chronic inflammation suggests that alterations in this tissue as the result of disease or environmental factors may influence the development and progression of various obesity-related pathologies. This study investigated WAT cell-specific responses to an organometal compound, trimethyltin (TMT), to determine possible contribution to induced inflammation. Methods Human primary mature adipocytes and macrophage differentiated THP-1 cells were cultured in TMT presence and relative toxicities and different adipokine levels were determined. The inflammatory response was examined in TMT presence for primary cells from obese ob/ob mice WAT, and after TMT injection in ob/ob mice. Results Both adipocytes and macrophages were resistant to cell death induced by TMT. However, adipocytes cultured in TMT presence showed increased expression of TNFα and IL-6, and modified leptin levels. In macrophage cultures, TMT also increased TNFα and IL-6, while MCP-1 and MIP-1α were decreased. In vivo, a single injection of TMT in ob/ob mice, elevated TNFα, MIP-1α and adiponectin in WAT. Conclusions Elevation of the inflammatory related products can be induced by chemical exposure in adipocytes and macrophages, as well as murine WAT. These data suggest that numerous factors, including a systemic chemical exposure, can induce an inflammatory response from the WAT. Furthermore, when characterizing both chemical-induced toxicity and the progression of the chronic inflammation associated with elevated WAT content, such responses in this target tissue should be taken into consideration.

The intravenous anesthetic propofol inhibits lipopolysaccharide-induced hypoxia-inducible factor 1 activation and suppresses the glucose metabolism in macrophages

Hypoxia-inducible factor 1 (HIF-1) is a master transcription factor of hypoxia-induced gene expression. Anesthetics and perioperative drugs have been reported to affect HIF-1 activity. However, the effect of propofol on HIF-1 activity is not well documented. In this study, we investigated the effect of propofol on HIF-1 activation using macrophage-differentiated THP-1 cells. Cells were exposed to lipopolysaccharide (LPS) under 20 or 1% O(2) conditions with or without propofol treatment. The cell lysate was subjected to Western blot analysis using anti-HIF-1alpha and HIF-1beta antibodies. HIF-1-dependent gene expression was investigated by quantitative real-time reverse-transcriptase PCR analysis and luciferase assay. The amount of cellular lactate and ATP was assayed. Propofol suppressed HIF-1alpha protein accumulation induced by LPS, but not by hypoxia in the THP-1 cells in a dose-dependent manner by inhibiting the neo-synthesis of HIF-1alpha protein. Induction of the HIF-1 downstream gene expression including glucose transporter 1, enolase 1, lactate dehydrogenase A, pyruvate dehydrogenase kinase-1 and vascular endothelial growth factor was inhibited by propofol. Propofol suppressed LPS-induced lactate accumulation and ATP content in THP-1 cells. Our experimental results indicate that propofol inhibits HIF-1 activation and downstream gene expression induced by LPS and suppressed HIF-1-dependent glucose metabolic reprogramming. HIF-1 suppression by propofol in macrophages may explain molecular mechanisms behind the inhibitory effect of propofol on cellular inflammatory responses.

The intravenous anesthetics barbiturates inhibit hypoxia-inducible factor 1 activation

Hypoxia-inducible factor 1 (HIF-1) is a master transcription factor of hypoxia-induced gene expression. Anesthetics and perioperative drugs have been reported to affect HIF-1 activity. However, the effect of barbiturates on HIF-1 activity has not been reported. In this study, we investigated the effect of thiopental and thiamylal on HIF-1 activity using the neuronal SH-SY5Y cells, the non-neuronal HEK293 cells, and the macrophage-differentiated THP-1 cells. Cells were exposed to 20% or 1% O 2 conditions with or without thiopental or thiamylal treatment. The cell lysate were subjected to Western blot analysis using anti-HIF-1α and -HIF-1β antibodies. HIF-1-dependent gene expression was investigated by semi-quantitative real-time RT-PCR and luciferase assay. Hydroxylation of HIF-1α protein was evaluated by in vitro pulldown assay using recombinant protein. Both thiopental and thiamylal reversibly suppressed hypoxia-induced HIF-1 activation in the neuronal and the non-neuronal cells in a dose-dependent manner. Moreover, the barbiturates inhibited lipopolysaccharide-induced HIF-1α expression in THP-1 cells. The HIF-1-downstream gene expression was also inhibited by the barbiturates. HIFα-hydroxylases activity and HIF-1α stability were not affected but the HIF-1α protein neosynthesis was inhibited by the barbiturates. Our experimental results indicate that barbiturates inhibit induced HIF-1 activation and downstream genes expression.