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In vitro approaches to address key steps of chemical-induced skin sensitization have been developed, but there is uncertainty how keratinocytes, which play a crucial role not only regarding xenobiotic metabolism but also skin inflammation, impact on a chemical's potential and potency to activate dendritic cells. We investigated these aspects by coculturing THP-1 cells, as surrogate dendritic cells, with HaCaT keratinocytes. We tested our HaCaT/THP-1 model with a set of 14 sensitizers, containing 7 prohaptens, and 10 non-sensitizers. Compared to exposing THP-1 alone, coculturing resulted in up to 3.1-fold enhanced maximal CD86 and/or CD54 upregulation on THP-1, and improved concentration-dependency. All 14 sensitizers were found positive for CD86 and/or CD54 upregulation based on ∆ mean fluorescence intensity (MFI) ≥ 10 for CD86 and ∆MFI ≥ 50 for CD54. Only 1 of 10 non-sensitizers was false-positive. Remarkably, coculture with HaCaT keratinocytes improved the rank correlation of the estimated minimum chemical concentrations inducing a positive response in vitro with in vivo data on sensitization potency, especially for CD54 (Spearman: r = 0.739, p = 0.006; CD86: r = 0.571, p = 0.041). These promising data suggest that the coculture model has the potential to support the prediction of sensitization potency based on in vitro data.

Background Fargesin is a lignan from Magnolia fargesii , an oriental medicine used in the treatment of nasal congestion and sinusitis. The anti-inflammatory properties of this compound have not been fully elucidated yet. Purpose This study focused on assessing the anti-inflammatory effects of fargesin on phorbal ester (PMA)-stimulated THP-1 human monocytes, and the molecular mechanisms underlying them. Methods Cell viability was evaluated by MTS assay. Protein expression levels of inflammatory mediators were analyzed by Western blotting, ELISA, Immunofluorescence assay. mRNA levels were measured by Real-time PCR. Promoter activities were elucidated by Luciferase assay. Results It was found that pre-treatment with fargesin attenuated significantly the expression of two major inflammatory mediators, cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). Fargesin also inhibited the production of pro-inflammation cytokines (IL-1β, TNF-α) and chemokine (CCL-5). Besides, nuclear translocation of transcription factors nuclear factor-kappa B (NF-ĸB) and activator protein-1 (AP-1), which regulate multiple pro-inflammatory genes, was suppressed by fargesin in a PKC-dependent manner. Furthermore, among the mitogen-activated protein kinases (MAPKs), only c-Jun N-terminal kinase (JNK) was downregulated by fargesin in a PKC-dependent manner, and this reduction was involved in PMA-induced AP-1 and NF-ĸB nuclear translocation attenuation, demonstrated using a specific JNK inhibitor. Conclusion Taken together, our results found that fargesin exhibits anti-inflammation effects on THP-1 cells via suppression of PKC pathway including downstream JNK, nuclear factors AP-1 and NF-ĸB. These results suggest that fargesin has anti-inflammatory properties with potential applications in drug development against inflammatory disorders.

The levels of TLR/RLR gene expression and production of some cytokines were studied in monocytic THP-1 cell line during its differentiation to mature macrophage-like forms induced by phorbol 12-myristate 13-acetate (PMA) treatment for 1 and 5 days in vitro. For the first time, we have shown high induction levels for the genes that encode signaling immune receptors and transcription factors in response to PMA, as well as inhibitory effects of TLR3, TLR7/TLR8, TLR9-agonists in mature macrophages. The PMAactivated THP-1 macrophage-like cells secreted large quantitities of inflammatory IL-1β and TNFα cytokines into culture medium.

After publication of our article [1], we found that the second images in the first row in Figure 2a were inadvertently replaced with the first image in Figure 2a.

Hypertriglyceridemia induces atherosclerosis and accordingly is a major causative factor in cardiovascular diseases. Macrophages that develop into foam cells are a crucial component in the development of atherosclerosis. Monocytes can be differentiated into M1 or M2 macrophages. M1 macrophages promote inflammatory responses, whereas M2 macrophages exhibit anti-inflammatory activity. Recently, we found that triglyceride (TG)-treated THP-1 monocytes express a variety of macrophage-specific surface markers, indicating that TG treatment could trigger the differentiation of monocytes into macrophages. In this study, we investigated whether TG-induced macrophages express the M1 or the M2 macrophage phenotype. THP-1 cells were treated with various concentrations of TG for different times and the expression of M1- and M2-specific markers was evaluated by RT-PCR. We found increased expression of M1 markers (CD40, CD80, and CD86) in TG-treated THP-1 cells in a TG dose- and time-dependent manner. The expression of M2 markers (CD163, CD200R, and CD206) showed variable responses to TG treatment. Taken together, our results indicate that TG treatment triggers the differentiation of monocytes into M1 macrophages, rather than into M2 macrophages, suggesting that TG contributes to pro-inflammatory responses.

A recent study reported that Pleurotus ostreatus has the potential to be used as a β-glucan- based cream for supportive complementary therapy of atopic dermatitis. KH054 is a new herbal prescription consisting of P. ostreatus and Panax ginseng. The effects of atopic dermatitis-induced materials on the expression of cytokine genes in human monocytes (THP-1, EoL- 1) have been examined. Some reports demonstrated that P. ginseng augments the activity of natural killer cells, which plays an important role in innate immunity against infection and tumor development. Monocyte chemotactic protein 1 (MCP-1), interleukin (IL)-6, and IL-8 have important roles in mediating the infiltration of various cells into the skin of atopic dermatitis and psoriasis. The present study investigated whether KH054 on induced IL-6, IL-8, and MCP-1 secretion by house dust mite (Dermatophagoides pteronissinus) in THP-1 (human acute monocytic leukemia) and EoL-1 (Human eosinophilic leukemia) cell. D. pteronissinus functions in the pathogenesis of allergic diseases, including atopic dermatitis and asthma. The inhibitory effect of KH054 on the induction of IL-6, IL-8, and MCP-1 secretion by D. pteronissinus extract in THP-1 and EoL-1 cells was examined. KH054 potently suppressed the elevated production of IL-6 and IL-8 induced by D. pteronissinus treatment in THP-1 and EoL-1 cells. Based on the present results, KH054 may be useful for developing functional foods to treat atopic dermatitis.

Thorough characterization of toxic effects of nanoparticles (NP) is desirable due to the increasing risk of potential environmental contamination by NP. In the current study, we combined three recently developed proteomics approaches to assess the effects of Au, CuO, and CdTe NP on the innate immune system. The human monocyte cell line THP-1 was employed as a model. The anticancer drugs camptothecin and doxorubicin were used as positive controls for cell death, and lipopolysaccharide was chosen as a positive control for proinflammatory activation. Despite equivalent overall toxicity effect (50 ± 10% dead cells), the three NP induced distinctly different proteomics signatures, with the strongest effect being induced by CdTe NP, followed by CuO and gold NP. The CdTe toxicity mechanism involves down-regulation of topoisomerases. The effect of CuO NP is most reminiscent of oxidative stress and involves up-regulation of proteins involved in heat response. The gold NP induced up-regulation of the inflammatory mediator, NF-κB, and its inhibitor TIPE2 was identified as a direct target of gold NP. Furthermore, gold NP triggered activation of NF-κB as evidenced by phosphorylation of the p65 subunit. Overall, the combined proteomics approach described here can be used to characterize the effects of NP on immune cells.

We have recently proposed that the interaction between food components and nanoparticles (NPs) should be considered when evaluating the toxicity of NPs. In the present study, we used THP-1 differentiated macrophages as a model for immune cells and investigated the combined toxicity of low levels of palmitate (PA; 10 or 50 μM) and ZnO NPs. The results showed that PA especially at 50 μM changed the size, Zeta potential and UV–vis spectra of ZnO NPs, indicating a possible coating effect. Up to 32 μg/mL ZnO NPs did not significantly affect mitochondrial activity, intracellular reactive oxygen species (ROS) or release of interleukin 6 (IL-6), but significantly impaired lysosomal function as assessed by neutral red uptake assay and acridine orange staining. The presence of 50 μM PA, but not 10 μM PA, further promoted the toxic effects of ZnO NPs to lysosomes but did not significantly affect other endpoints. In addition, ZnO NPs dose-dependently increased intracellular Zn ions in THP-1 macrophages, which was not significantly affected by PA. Taken together, the results of the present study showed a combined toxicity of low levels of PA and ZnO NPs especially to lysosomes in THP-1 macrophages.

Background Javamide-I and-II are phenolic amide compounds found in Coffea sp. Previous study suggested that javamide-II may be a potent compound with Sirt1/2 inhibition activity. Purpose However, the effects of javamide-I and the its O-methyl ester on Sirt inhibition, p53 acetylation and cell death have not been investigated. Methods The isolation and synthesis of javamide-I and its O-methyl ester analogue were confirmed by NMR. Their potential effects on Sirt1/2/3, p53 acetylation and cell death were examined using sirt assay, silico analysis, Western blot, caspase 3/7 and apoptotic assay methods. Results Javamide-I and its O-methyl ester demonstrated a similar inhibition pattern; Sirt1 (IC 50 of 19 µM) better than Sirt2 (IC 50 of 104 µM) and Sirt3 (IC 50 of 160 µM). However, javamide-I and its O-methyl ester were found to inhibit Sirt1 better than Sirt2, which is different from javamide-II able to inhibit Sirt2 stronger than Sirt1. In silico analysis, javamide-I and its O-methyl ester showed a competitive binding pattern against NAD +, which was also supported by the kinetic analysis with K i = 20.1 µM for javamide-I and 19.5 µM for the O-methyl ester. However, the O-methyl ester increased p53 acetylation better than javamide-I in monocytic THP-1 cells. Since caspase 3/7 activation is often followed by the p53 activation, we investigated their effects on caspase 3/7, and found that O-methyl ester again activated caspase 3/7 greater than javamide-I in the cells, eventually leading to apoptotic cell death. Conclusion These data suggest that the O-methyl modification in javamide-I may play a critical role in increasing p53 acetylation, activating caspase, and eventually inducing the THP-1 cell death.

BACKGROUND This study was aimed to reveal the role of miR-149-5p in acute myeloid leukemia (AML) cells apoptosis and the possible mechanism involved. MATERIAL AND METHODS The expression of miR-149-5p in leukemia cell lines, as well as the blood and bone marrow (BM) samples from leukemia patients, were monitored by reverse-transcription polymerase chain reaction (RT-PCR). AML cell line THP-1 was transfected with miR-149-5p mimic or inhibitor, and then cell apoptosis was determined using the APO Percentage assay kit. The target of miR-149-5p was predicted by using the microRNA.org database, and verified by RT-PCR, Western blot, and Dual-Luciferase reporter assays. Further, small interfering RNA (siRNA) against the target gene was co-transfected with miR-149-5p inhibitor, and then the cell apoptosis and the expression of apoptosis-related proteins were assessed. RESULTS MiR-149-5p was significantly up-regulated in leukemia cell lines and samples from leukemia patients (P

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.

Sepsis is characterized by systemic activation of inflammatory and coagulation pathways in response to infection. Recently, it was demonstrated that histones released into the circulation by dying/activated cells may contribute to sepsis pathology. Although the ability of extracellular histones to modulate the procoagulant activities of several cell types has been investigated, the influence of histones on the hemostatic functions of circulating monocytes is unknown. To address this, we investigated the ability of histones to modulate the procoagulant potential of THP-1 cells and peripheral blood monocytes, and examined the effects of plasmas obtained from septic patients to induce a procoagulant phenotype on monocytic cells.Tissue factor (TF) activity assays were performed on histone-treated THP-1 cells and blood monocytes. Exposure of monocytic cells to histones resulted in increases in TF activity, TF antigen, and phosphatidylserine exposure. Histones modulate the procoagulant activity via engagement of Toll-like receptors 2 and 4, and this effect was abrogated with inhibitory antibodies. Increased TF activity of histone-treated cells corresponded to enhanced thrombin generation in plasma determined by calibrated automated thrombography. Finally, TF activity was increased on monocytes exposed to plasma from septic patients, an effect that was attenuated in plasma from patients receiving unfractionated heparin (UFH).Our studies suggest that increased levels of extracellular histones found in sepsis contribute to dysregulated coagulation by increasing TF activity of monocytes. These procoagulant effects can be partially ameliorated in sepsis patients receiving UFH, thereby identifying extracellular histones as a potential therapeutic target for sepsis treatment.

Chemokine CCL14 is inactive in its proform. Here, we show that inflammation- and cancer-associated kallikrein-related peptidases KLK5 and KLK8 remove the N-terminal eight amino acids from the proform thereby converting CCL14 to its active state. Activity of the chemokine is demonstrated by migration of myeloid cells expressing relevant receptors.

The THP-1 cell line is broadly used as a model for acute myeloid leukemia (AML) with MLL fusion and to study monocyte differentiation and function. We studied THP-1 cells obtained from two major biorepositories. The two cell lines were closely related with a percentage match of short tandem repeat (STR) profiles ranging from 93.75% to 100%, depending on the algorithm used. Nevertheless, we found that the two cell lines presented discordant HLA type, cytogenetic aberrations and AML-related gene expression (including critical targets of MLL fusion). These discrepancies resulted mainly from loss of heterozygosity (LOH) involving five chromosomal regions. In view of their aberrant expression of key "leukemia" genes (e.g., LIN28B, MEIS1 and SPARC), we argue that one of the THP-1 cell lines may not be a reliable model for studying leukemia. Their defective expression of HLA molecules and abnormal adhesion properties is also a caveat for studies of antigen presentation. In a more general perspective, our findings show that seemingly minor discrepancies in STR profiles among cell lines may be the sign of major genetic drift, of sufficient magnitude to affect the reliability of cell line-based research.

The human innate immune response to the pore-forming toxin of Vibrio cholerae VCC, is currently under study. Here, in vitro studies on a human macrophage cell line (THP-1), helped explore the activated pathways involved on the onset the innate immune response towards the cytotoxin. The secreted monomeric 65 KDa form interacts with mature macrophages in pg/ml concentrations, determined by dose response experiments after treatments under 1 h. Non vacuolating concentrations (pg/ml) were applied to the cells; immunoblots revealed activation of MAPKs: early overexpression of p38 and ERK. Cell lysis by release of lactate dehydrogenase (LDH) was not apparent in the first hour, nonetheless it was positive after 24 h. Finally, to discern whether the VCC stimulates transcriptional activators via MAPKs pathway, NF-κB and AP-1 were studied by real time quantitation. Increased expression of p50 (NF-κB), cJun and cFos (AP-1) was observed. Given that NF-κB is the transcription factor initiating inflammation of innate immune response and in turn, AP-1 is responsible for cell surviving response, results from this study lead us to conclude that VCC in vitro treatments, induce a pro-inflammatory and a surviving response, in less than one hour on activated macrophages.

Recent studies have shown that transcriptomic analysis of blood samples taken from patients with acute Ebola virus disease (EVD) during the 2013&ndash, 2016 West African outbreak was suggestive that a severe inflammatory response took place in acutely ill patients. The significant knowledge gained from studying the Makona variant, a cause of the largest known EVD outbreak, may be applicable to other species of ebolavirus, and other variants of the Ebola virus (EBOV) species. To investigate the ability of Makona to initiate an inflammatory response in human macrophages and characterise the host response in a similar manner to previously characterised EBOV variants, the human monocytic cell line THP-1 was differentiated into macrophage-like cells and infected with Makona. RNA-Seq and quantitative proteomics were used to identify and quantify host mRNA and protein abundance during infection. Data from infection with Reston virus (RESTV) were used as comparators to investigate changes that may be specific to, or enhanced in, Makona infection in relation to a less pathogenic species of ebolavirus.. This study found demonstrable induction of the inflammatory response, and increase in the activation state of THP-1 macrophages infected with Makona. NF&kappa, B and inflammation-associated transcripts displayed significant changes in abundance, reflective of what was observed in human patients during the 2013&ndash, 2016 EBOV outbreak in West Africa, and demonstrated that transcriptomic changes found in Makona-infected cells were similar to that observed in Reston virus infection and that have been described in previous studies of other variants of EBOV.

Cyclin-dependent kinase 9 (CDK9), one crucial molecule in promoting the transition from transcription pausing to elongation, is a critical modulator of cell survival and death. However, the pathological function of CDK9 in bacterial inflammatory diseases has never been explored. CDK9 inhibition or knock-down attenuated Porphyromonas gingivalis-triggered inflammatory gene expression. Gene-expression microarray analysis of monocytes revealed that knock-down of CDK9 not only affected inflammatory responses, but also impacted cell death network, especially the receptor-interacting protein kinase 3 (RIPK3)-mixed lineage kinase domain-like (MLKL)-mediated necroptosis after P. gingivalis infection. Inhibition of CDK9 significantly decreased necroptosis with downregulation of both MLKL and phosphorylated MLKL. By regulating caspase-8 and cellular FLICE inhibitory protein (cFLIP), key molecules in regulating cell survival and death, CDK9 affected not only the classic RIPK1-RIPK3-mediated necroptosis, but also the alternate TIR-domain-containing adapter-inducing interferon-β-RIPK3-mediated necroptosis. CDK9 inhibition dampened pro-inflammatory gene production in the acute infection process in the subcutaneous chamber model in vivo. Moreover, CDK9 inhibition contributed to the decreased periodontal bone loss and inflammatory response induced by P. gingivalis in the periodontal micro-environment. In conclusion, by modulating the RIPK3-MLKL-mediated necroptosis, CDK9 inhibition provided a novel mechanism to impact the progress of bacterial infection in the periodontal milieu.

5-lipoxygenase (5-LO), coded by the ALOX5 gene, is expressed in leukocytes and catalyzes the formation of leukotrienes, pro-inflammatory lipid mediators. Leukotrienes are central to immune responses, but are also involved in inflammatory disorders and 5-LO expression is associated with leukemia stem cell survival. It is therefore important to understand mechanisms that control 5-LO expression. This study investigated the control of 5-LO expression and leukotriene biosynthesis following the maturation of human monocytic cells. MonoMac-1 (MM1) and THP-1 cells were incubated for up to 72 h with or without LPS and TGF-β. LPS, but not TGF-β, increased CD14 expression in both MM1 and THP-1 cells. Incubation with LPS (100 ng/ml) and TGF-β (1 ng/ml) synergistically increased the capacity of MM1 cells to produce 5-LO products from undetectable levels to 40±5 pmol/106 cells. 5-LO product biosynthesis in THP-1 cells increased 25-fold. A synergistic effect of LPS and TGF-β was measured with increases in 5-LO mRNA of 54- and 13-fold in MM1 and THP-1 cells, respectively. 5-LO protein expression increased significantly in both MM1 and THP-1 cells. ALOX5 promoter activity was significantly elevated >2-fold in both cell lines following LPS treatment, but TGF-β was without effect. The main 5-LO products were cysteinyl-leukotrienes, however LPS and TGF-β did not impact on the capacity of the cells to metabolize leukotriene A4. Overall, this study demonstrates that receptor-mediated stimulation of MM1 and THP-1 cells by LPS is associated with increased 5-LO expression. This represents a new mechanism by which leukotriene biosynthesis can be modulated by pathological agents.

Background The endocannabinoid (eCB) 2-arachidonoylglycerol (2-AG) is a known modulator of inflammation and few studies have addressed its influence on myeloid cells in the context of atherogenesis. However, the impact of 2-AG on endothelial cell function has not been studied before. Methods Endothelial repair was studied in two treatment groups of wildtype mice following electrical denudation of the common carotid artery at a length of 3000 μm. One group received the monoacylglycerol lipase (MAGL)-inhibitor JZL184 [5 mg/kg i.p.], which impairs 2-AG degradation and thus causes elevated 2-AG levels, the other group received vehicle. The residual endothelial gap at five days in either group was visualized by Evan's blue staining. In vitro, the effect of 2-AG on human coronary artery endothelial cell (HCAEC) viability was assessed by an XTT-based assay. Endothelial activation was studied by an adhesion assay of THP-1 monocytes to 2-AG-preconditioned HCAEC. HCAEC migration, ROS-production, expression of NADPH oxidases, and secretion of inflammatory cytokines were assessed by Boyden chamber, qPCR, and colorimetric assays. Results Treatment with JZL184 produced a significant increase in 2-AG levels and impaired reendothelialisation in wildtype mice following electrical injury of the common carotid artery. The residual denudation at 5 days yielded 2291±286 μm in JZL184-treated animals vs. 1505±223 μm in vehicle treated controls (n=18–19; p Conclusion Elevated 2-AG levels appear to hamper endothelial repair and to promote HCAEC activation and cell death. Our data suggest that besides its influence on myeloid cells, 2-AG is also adverse to endothelial integrity which might promote early atherosclerotic lesion formation. Thus, decreasing vascular 2-AG levels might represent a promising therapeutic strategy for the prevention of atherosclerosis and coronary heart disease.

Objectives The aim of this study is to evaluate the inhibitory effects of M. alba stem extract (MSE) on the expression of matrix metalloproteinases (MMP)-1, MMP-9, and tissue inhibitors of metalloproteinase (TIMP)-1 in Porphyromonas gingivalis lipopolysaccharide (LPS)-activated-acute monocytic leukemia cell line (THP-1). Materials and Methods THP-1 cells were treated with noncytotoxic concentrations of MSE combined with 1 µg/mL of P. gingivalis LPS. The mRNA levels of MMP-1, MMP-9, and TIMP-1 were evaluated via quantitative real-time polymerase chain reaction. The secreted proteins in the culture media were detected by enzyme-linked immunosorbent assay. The degradation of inhibitor of kappa B-alpha (IκBα) protein was tracked by Western blotting. Statistical Analysis Comparisons in experiments were analyzed with analysis of variance followed by Tukey honestly significant difference comparison test. ResultsTwenty and 40 µg/mL of MSE significantly downregulated MMP-1 and MMP-9 genes and protein expression but upregulated the gene expression of TIMP-1 (p < 0.05). P. gingivalis LPS induced degradation of IκBα, while addition of MSE (20 and 40 µg/mL) increased IκBα cytosolic levels. MSE was able to suppress the P. gingivalis LPS-induced MMPs expression and also increased the gene expression of TIMP-1 via the inhibition of the cytoplasmic IκBα degradation in THP-1 cells. Conclusions The present observations suggest that MSE exerted a positive effect on the regulatory mechanism between MMPs and TIMP, which is an important implication for the therapeutic potential of MSE in periodontitis.

Introduction The intercellular transfer of biologically active molecules in extracellular vesicles (EVs) has recently been discovered as an important mechanism, which regulates cardiovascular health and disease. In the context of atherosclerosis, endothelial cell-derived EVs have been shown to modulate the phenotype of EV recipient cells in a relevant manner. Under pro-atherogenic conditions e.g. hyperglycemia, the export of numerous signal molecules in EVs is altered and so are EV-dependent effects on recipient cells. While the effect of endothelial-cell derived EVs on other endothelial cells and vascular smooth muscle cells is well characterized, little is known about the vesicle-based interaction of endothelial cells and monocytes under pro-atherogenic conditions. This is particularly relevant as monocytes are crucial modulators of vascular regeneration and inflammation. Our aim was therefore to investigate, whether EVs from endothelial cells have a significant effect on the differentiation and polarization of monocytes and how this process is affected by pathologic conditions as mentioned above. Methods and results Human Coronary Arterial Endothelial Cells (HCAECs) were cultured in high-Glucose-medium (30 mM) for 72h. PBS was used as a control. Large EVs were isolated from the culture supernatant by differential centrifugation (1 x 1500 g / 15 min + 2 x 2ehz748.0909 g / 40 min). The harvested large EVs were characterized by Immunoblotting, Nanoparticle Tracking Analysis as well as Transmission electron microscopy and were shown to be mostly between 80 and 500 nm in size. Specific surface markers including Annexin V and Flotillin-1 were highly enriched in the isolated EVs. The EVs were used for co-culture with THP-1 cells with and without previous phorbol-12-myristate-13-acetate (PMA) stimulation. After 4h as well as after 24 h of incubation with EVS, total RNA was isolated from the THP-1 cells and qPCR was performed to assess polarization towards M1 by TNF-α gene expression or M2 by IL-10 expression. While EV treatment of THP-1 cells without previous PMA-stimulation showed no measurable effect, a significant decrease in the expression of TNF-α was detected after 4 h of treatment from Glucose injured HCAECs. Similar results were obtained without glucose stimulation, the most significant reduction of TNF-α expression however was seen at 24 h. In regard to IL-10 no significant expression changes were detected in EV treated THP-1 cells. Conclusion We showed that glucose injury does not relevantly affect vesicle release or size. Furthermore, endothelial cell derived EVs cause a reduction of TNF-α expression, which indicates a polarization towards an M2 macrophage phenotype, irrespective of prior hyperglycaemic injury. As the M2 phenotype has been described as pro-regenerative, we conclude that endothelial cell derived EVs can exert a protective function during the invasion of monocytes in cardiovascular disease and remodeling.

Glycogen synthase kinase-3 (GSK-3) is a multitasking protein kinase that regulates numerous critical cellular functions. Not surprisingly, elevated GSK-3 activity has been implicated in a host of diseases including pathological inflammation, diabetes, cancer, arthritis, asthma, bipolar disorder, and Alzheimer’s. Therefore, reagents that inhibit GSK-3 activity provide a means to investigate the role of GSK-3 in cellular physiology and pathophysiology and could become valuable therapeutics. Finding a potent inhibitor of GSK-3 that can selectively target this kinase, among over 500 protein kinases in the human genome, is a significant challenge. Thus there remains a critical need for the identification of selective inhibitors of GSK-3. In this work, we introduce a novel small organic compound, namely COB-187, which exhibits potent and highly selective inhibition of GSK-3. Specifically, this study 1) utilized a molecular screen of 414 kinase assays, representing 404 unique kinases, to reveal that COB-187 is a highly potent and selective inhibitor of GSK-3; 2) utilized a cellular assay to reveal that COB-187 decreases the phosphorylation of canonical GSK-3 substrates indicating that COB-187 inhibits cellular GSK-3 activity; and 3) reveals that a close isomer of COB-187 is also a selective and potent inhibitor of GSK-3. Taken together, these results demonstrate that we have discovered a region of chemical design space that contains novel GSK-3 inhibitors. These inhibitors will help to elucidate the intricate function of GSK-3 and can serve as a starting point for the development of potential therapeutics for diseases that involve aberrant GSK-3 activity.

After over a decade of nanosafety research, it is indisputable that the vast majority of nano-sized particles induce a plethora of adverse cellular responses - the severity of which is linked to the material's physicochemical properties. Differentiated THP-1 cells were previously exposed for 6 h and 24 h to silver, titanium dioxide, and zinc oxide nanoparticles at the maximum molar concentration at which no more than 15% cellular cytotoxicity was observed. All three nanoparticles differed in extent of induction of biological pathways corresponding to immune response signaling and metal ion homeostasis. In this study, we integrated gene and miRNA expression profiles from the same cells to propose miRNA biomarkers of adverse exposure to metal-based nanoparticles. We employed RNA sequencing together with a quantitative strategy that also enables analysis of the overlooked repertoire of length and sequence miRNA variants called isomiRs. Whilst only modest changes in expression were observed within the first 6 h of exposure, the miRNA/isomiR (miR) profiles of each nanoparticle were unique. Via canonical correlation and pathway enrichment analyses, we identified a co-regulated miR-mRNA cluster, predicted to be highly relevant for cellular response to metal ion homeostasis. These miRs were annotated to be canonical or variant isoforms of hsa-miR-142-5p, -342-3p, -5100, -6087, -6894-3p, and -7704. Hsa-miR-5100 was differentially expressed in response to each nanoparticle in both the 6 h and 24 h exposures. Taken together, this co-regulated miR-mRNA cluster could represent potential biomarkers of sub-toxic metal-based nanoparticle exposure.

The present study aimed to explore the mechanisms through which microRNA (miR)‑20a may be involved in the differentiation of THP‑1 human acute monocytic leukemia cells into osteoclasts. THP‑1 cells were differentiated into macrophages (osteoclast precursors) and subsequently into osteoclast cells. The expression levels of miR‑20a in THP‑1 cells were significantly reduced in a time‑dependent manner during phorbol‑12‑myristate‑13‑acetate (PMA), macrophage colony‑stimulating factor (M‑CSF) and receptor activator of nuclear factor‑κB ligand RANKL‑induced osteoclastogenesis. Following transfection with a miR‑20a mimics, the levels of miR‑20a in PMA‑treated THP‑1 cells increased more than 40‑fold as compared with expression in the control cells. In addition, the overexpression of miR‑20a inhibited proliferation, initiated S phase cell cycle arrest and induced apoptosis of PMA‑treated THP‑1 cells. Additionally, miR‑20a mimics treatment notably decreased the levels of tartrate‑resistant acid phosphatase, nuclear factor of activated T‑cells, cytoplasmic 1 and peroxisome proliferator‑activated receptor γ (PPARγ) during THP‑1 cell further differentiation progress. In summary, miR‑20a may negatively regulate the proliferation and osteoclastogenesis of THP‑1 cells during its osteoclast differentiation progress by downregulating PPARγ.

Background Iron overload is a major health concern for transfusion-dependent patients. Repeated transfusions result in the loading of large amounts of haem-derived iron on macrophages, in turn, inducing cell death. We previously demonstrated that haemin-induced cell death in human monocytic THP-1 cells is consistent with ferroptosis, an iron-dependent cell death regulation mechanism. However, direct measurement of iron after haemin treatment has not yet been conducted. In this study, we measured intracellular non-haem iron concentration and haem oxygenase levels after haemin treatment. Material and methods Human monocytic THP-1 cells were treated with haemin, and the cell lysate was prepared. Non-haem iron concentration of the cell lysate was measured using the Nitroso-PSAP method. Expression of haem oxygenase-1 (HO-1) and haem oxygenase-2 (HO-2) was quantified by western blotting. Results We measured intracellular non-haem iron and the expression of haem oxygenases post-haemin treatment. Concentration of non-haem iron post-haemin treatment increased dependently with time and dose. HO-1 expression was detected 4 h after haemin treatment, whereas HO-2 expression was constitutive. Discussion Increase in non-haem iron prior to induction of HO-1 expression suggests the involvement of HO-2 in haem-induced cytotoxicity. (184 words)

Cigarette smoking and tuberculosis are a significant cause of death worldwide. Several epidemiological studies have demonstrated cigarette smoking is a risk factor for tuberculosis. Electronic cigarettes have recently appeared as a healthier alternative to conventional smoking, although their impact in tuberculosis is not well understood. The aim of this study was to explore the effect of electronic cigarettes in phagocytosis of Mycobacterium tuberculosis and cytokines production. In vitro infection was carried out by exposing THP-1 macrophages to four electronic vapor extracts and the intracellular burden of M. tuberculosis was determined. The percentage of infection was evaluated by confocal microscopy and the cytokine production by Luminex. A reduction of intracellular M. tuberculosis burden in THP- 1 macrophages was found after its exposure to electronic vapor extract; the same trend was observed by confocal microscopy when Mycobacterium bovis BCG-GFP strain was used. Electronic cigarettes stimulate a pro-inflammatory cytokine response. We conclude that electronic cigarettes impair the phagocytic function and the cytokine response to M. tuberculosis.

When infecting a human host, Corynebacterium diphtheriae and Corynebacterium ulcerans are able to impair macrophage maturation and induce cell death. However, the underlying molecular mechanisms are not well understood. As a framework for this project, a combination of fluorescence microscopy, cytotoxicity assays, live cell imaging, and fluorescence-activated cell sorting was applied to understand the pathogenicity of two Corynebacterium strains isolated from fatal cases of systemic infections. The results showed a clear cytotoxic effect of the bacteria. The observed survival of the pathogens in macrophages and, subsequent, necrotic lysis of cells may be mechanisms explaining dissemination of C. diphtheriae and C. ulcerans to distant organs in the body.

GATA2, a zinc finger transcription factor predominantly expressed in hematopoietic cells, acts as an essential regulator of hematopoietic stem cell generation, survival and functionality. Loss and gain of GATA2 expression has been implicated in myelodysplastic syndrome and acute myeloid leukemia (AML) yet the precise biological impact of GATA2 expression on human AML cell fate decisions remains ambiguous. Herein, we performed large-scale bioinformatics that demonstrated relatively frequent GATA2 overexpression in AML patients as well as select human AML (or AML-like) cell lines. By using shRNAi to target GATA2 in these AML cell lines, and an AML cell line expressing normal levels of GATA2, we found that inhibition of GATA2 caused attenuated cell proliferation and enhanced apoptosis exclusively in AML cell lines that overexpress GATA2. We proceeded to pharmacologically inhibit GATA2 in concert with AML chemotherapeutics and found this augmented cell killing in AML cell lines that overexpress GATA2, but not in an AML cell line expressing normal levels of GATA2. These data indicate that inhibition of GATA2 enhances chemotherapy-mediated apoptosis in human AML cells overexpressing GATA2. Thus, we define novel insights into the oncogenic role of GATA2 in human AML cells and suggest the potential utilization of transient GATA2 therapeutic targeting in AML.

Chromoblastomycosis is a chronic, progressive fungal disease of the skin and subcutaneous tissue caused by a unique group of dematiaceous fungi. Fonsecaea monophora, a new species distinct from Fonsecaea pedrosoi strains, is the main pathogen responsible for chromoblastomycosis in south China. Macrophages can be polarized into two categories: classically activated and alternatively activated. Little is known about the relationship between F. monophora and macrophage polarization. This study aimed to study the effect of F. monophora on the polarization of THP-1 cells to macrophages. We established coculture systems of F. monophora and THP-1-derived macrophages in different activation states. F. monophora enhanced the phagocytosis by macrophages in the initially activated state and weakened the phagocytosis by classically activated macrophages without affecting that by alternatively activated macrophages. Classically activated macrophages had the strongest killing effect on F. monophora, while the initially activated macrophages had the weakest. The pathogen could not be rapidly cleared by any type of macrophage. F. monophora promoted the expression of proinflammatory cytokines and inhibited that of anti-inflammatory cytokines. F. monophora promoted the polarization of THP-1 cells to classically activated macrophages and inhibited that of THP-1 cells to alternatively activated macrophages.

Leflunomide (LFD) is an Aryl hydrocarbon receptor (AhR) agonist and immunomodulatory drug with several side effects. Niosomes are novel drug delivery systems used to reduce the unfavorable effects of drugs by enhancing their bioavailability, controlling their release and targeting specific sites.Here, we prepared niosomal formulations of LFD, evaluated their properties and delivered to THP-1 monocytic cells to study the activation and nuclear translocation of AhR.Four types of non-ionic surfactants were utilized to formulate niosomes by thin film hydration (TFH) method. Entrapment efficiency (EE %) of niosomes were quantified and dynamic light scattering (DLS) was performed. Transmission electron microscopy (TEM) was used to identify the morphology of LFD niosomes. Dialysis method was used to measure LFD release rate. MTS assay was adopted to examine the viability of the cells upon each treatment. The nuclear transfer of AhR was investigated by Immunocytochemistry (ICC). The mRNA expression of IL1β and CYP1A1 were evaluated using quantitative RT-PCR.Span 60: cholesterol (1:1) showed the highest EE% (70.00 ± 6.24), largest particles (419.00 ± 4.16 nm) and the best uniformity with the lowest PDI (0.291 ± 0.007). TEM micrographs of Span 60 (1:1) nanoparticles showed conventional spherical vesicles with internal aqueous spaces. The release rate of LFD from Span 60 (1:1) vesicles was slower. Although the viability of LFD niosome-treated THP-1 cells was decreased, they were associated with lower cytotoxic effects compared with the free LFD counterparts. Both free and niosomal LFD treatments intensified the nuclear translocation of AhR. The mRNA expression of CYP1A1 was overexpressed while IL1β was downregulated in both free and niosomal LFD treated combinations.LFD encapsulation in Span 60: cholesterol (1:1) niosomal formulation could be introduced as a suitable vehicle of transferring LFD to THP-1 cells, with minimal cytotoxic effects, enhancing the AhR nuclear translocation and activation and inducing immunomodulatory properties. Graphical abstract The Graphical abstract; it demonstrates the workflow of the study and summary of results in brief.

Context: We reported that D-4F, an apolipoprotein A-I (Apo A-I) mimetic polypeptide with 18 d-amino acids, suppressed IL-4 induced macrophage alternative activation and TGF-β1 expression in phorbol 12-myristate 13-acetate (PMA) treated human acute monocytic leukemia cells (THP-1). Objective: Macrophage alternative activation, TGF-β1 and epithelial-mesenchymal transition (EMT) are intensively involved in pulmonary fibrosis. Recent studies demonstrated that Apo A-I resolved established pulmonary fibrotic nodules, and D-4F inhibited TGF-β1 induced EMT in alveolar cells. Therefore, this study evaluated the effects of D-4F on IL-4 induced macrophage alternative activation and TGF-β1 expression. Materials and methods: THP-1 cells were simulated with PMA (100 ng/mL) for 48 h and treated with medium control, IL-4 (20 ng/mL) alone, or IL-4 (20 ng/mL) in the presence of D-4F (1, 5, and 10 μg/mL) for 24 and 48 h. Flow cytometry, RT-PCR and ELISA evaluations were performed to investigate the subsequent effects of D-4F. Results: Compared to stimulation with IL-4 alone, 1, 5, and 10 μg/mL of D-4F reduced alternative activation by 45.38%, 59.98%, and 60.10%, increased TNF-α mRNA levels by 8%, 11%, and 16% and decreased TGF-β1 mRNA levels by 21%, 37%, and 39%, respectively (all p ≤ 0.05). In addition, TNF-α protein levels increased from 388 pg/mL (IL-4 alone) to 429, 475, and 487 pg/mL (1, 5, and 10 μg/mL D-4F), while TGF-β1 protein levels dropped from 27.01 pg/mL (IL-4 alone) to 19.15, 12.27, and 10.47 pg/mL (1, 5, and 10 μg/mL D-4F). Conclusion: D-4F suppressed IL-4 induced macrophage alternative activation and pro-fibrotic TGF-β1 expression.

This protocol details the experimental procedure for performing the comet assay, a very sensitive DNA break assay based on single cell gel electrophoresis.The analysis of DNA strand breaks, both single- and double-strand breaks (SSBs and DSBs, respectively), was performed in immune responsive cells. The cell line used was the human monocytic cell line THP-1, an adherent cell type with many known applications in in vitro studies of innate immunity. The comet assay is a robust procedure that allows the accurate and reproducible quantification of DNA damage. Here we describe not only the comet assay step-by-step protocol, but also some important aspects related to troubleshooting.

30 Páginas.-- 5 Figuras.-- 2 Tablas, Three olive modified pectin extracts have been produced by heat and acid treatment of the major by-product of olive oil production. Their effect on proliferation of the colon carcinoma Caco-2 and the leukemia monocytic THP-1 cell lines has been studied in order to determine possible anti-tumor properties. All extracts inhibited proliferation at some of the concentrations ranging from 1 to 10 mg ml−1. Interestingly none of the extracts inhibited the growth of confluent Caco-2 cells, showing the specificity of the antiproliferative effect for the transformed Caco-2 phenotype. All the extracts inhibited agglutination of red blood cells by galectin-3, a lectin involved in tumor growth, metastasis, and immune cell regulation that has been proposed as a mediator of the anti-tumor effects of modified pectins. In addition, activation of caspase-3 in THP-1 cells indicates that treatment with the pectin-rich extracts triggers apoptosis. These results point to a possible use as health-promoting food ingredients or supplements., This research was supported by the Spanish Ministry of Economy, Industry and Competitiveness and co-funded by a European Social Fund (ESF) (Ramon y Cajal Programme Grant 2012-10456; AGL2013-48291-R; and AGL2016-79088-R). A. Bermúdez-Oria received funding from the Spanish FPI program (MEIC) (BES-2014-068508).

Chronic inflammatory conditions can negatively impact intestinal barrier function and affect the epithelium's interaction with nano-sized materials. We demonstrate the application of a Caco-2/THP-1 co-culture mimicking the intestine in healthy (i.e. stable) or inflamed state in nanotoxicological research. The co-cultures were exposed to non-toxic concentrations of silver nanoparticles (AgNPs) or silver nitrate (AgNO3) for 24 h. The barrier integrity and cytokine release as well as necrotic and apoptotic cell death were investigated. AgNPs and AgNO3 most strongly affected the inflamed co-culture. Higher concentrations of AgNPs induced a significant increase in barrier integrity in the inflamed but not the stable co-culture. Necrotic and apoptotic cell death was detected in both conditions but were significantly more pronounced in the inflamed condition. The exposure to AgNO3 affected barrier integrity in all experimental set-ups, but caused nuclear condensation only in the Caco-2 monoculture and the inflamed co-culture. AgNPs reduced the release of monocyte chemoattractant protein-1 in the stable model. Clear differences were observed in the effects of AgNPs and AgNO3 in relation to the model's health status. The results suggest an increased vulnerability of the inflamed epithelial barrier towards AgNPs underlining the importance to consider the intestinal health status in the safety assessment of nanomaterials., Highlights • In inflamed conditions, Caco-2 cells are more susceptible to AgNP-induced effects than cells in homeostatic co-cultures. • Inflammation-like processes enhance the occurrence of AgNP-induced necrosis and apoptosis. • AgNPs might influence gut homeostasis by inhibiting monocyte chemoattractant protein-1.

S100A8 is a damage-associated molecular pattern protein released by monocytes, playing a decisive role in the development of inflammation. Nonresolving inflammation is viewed as a driving force in tumorigenesis, and its role in tumor immune escape also attracted attentions. PD-1/PD-L1 axis is a critical determinant of physiological immune homeostasis, and anti–PD-1 or PD-L1 therapy has becoming the most exciting field of oncology. Multiple regulation mechanisms have been contributed to PD-L1 expression modulation including inflammatory mediators. In this study we reported that S100A8 significantly induced PD-L1 expression in monocytes/macrophages but not in tumor cells. S100A8 induced PD-L1 transcription through the TLR4 receptor and multiple crucial pathways of inflammation process. S100A8 modulated the histone modification of the PD-L1 promoter in monocytes/macrophages. S100A8-pretreated macrophages had immunosuppressive function and attenuated the antitumor ability of CTLs both in vitro and in vivo. A highly positive correlation existed between S100A8 expression and PD-L1 expression in human cancer specimens. To our knowledge, our study uncovers a novel molecular mechanism for regulating PD-L1 transcription by an inflammatory mediator S100A8, and reveals the importance of comprehensive understanding the role of inflammation in tumorigenesis as well as in tumor immune escape.

Polyphenols as typical food components can influence the colloidal properties and internalization of nanomaterials (NMs) into mammalian cells. Recently, we found that 3-hydroxyflavone (H3) promoted intracellular Zn ions in ZnO nanoparticle (NP) exposed Caco-2 and HepG2 cells. However, it is unclear if H3 could affect the internalization of metal-based NMs with different morphologies. This study investigated the influence of H3 on colloidal aspects of Ag NPs and Ag nanoflakes (NFs) as well as the internalization of Ag NMs into THP-1 macrophages. 3-Hydroxyflavone at 50 μM promoted the solubility and altered hydrodynamic size, polydispersity index, and ζ potential of Ag NPs and Ag NFs, which indicated that H3 could affect the colloidal stability of Ag NMs. Only H3 but not Ag NMs significantly decreased mitochondrial activities of THP-1 macrophages. The internalization of Ag NMs was markedly increased due to the presence of H3. 3-Hydroxyflavone also exhibited antioxidative properties as it reduced intracellular reactive oxygen species and promoted the activities of ABC transporters as it reduced retention of Calcein in Ag NM-exposed THP-1 macrophages. We concluded that H3 promoted the internalization of Ag NMs into macrophages probably by altering the colloidal stability of Ag NMs and consequently NM-macrophage interactions.

BACKGROUND This study was designed to evaluate the effects of micro-RNA-16 (miR-16)-regulated expression of myeloblastosis oncogene (MYB) on the differentiation of acute leukemia cells, the expressions of miR-16 and MYB mRNA, and protein in differently differentiated leukemia cells were detected by real-time PCR and western blot. METHODS 1,25-Dihydroxyvitamin D3 (1,25 D3) induced monocytic differentiation of HL60 cells, and the resulting changes in miR-16 and MYB expressions were detected. Morphology of the cells induced by 1,25 D3, after being transfection with miR-16 mimics, was observed by Wright-Giemsa staining. The expression of mononuclear cell surface marker CD14 was detected by flow cytometry. RESULTS Minimum miR-16 was expressed in early-differentiation KG-1a cells, while late-differentiation U937 and THP-1 cells had higher expressions (p < 0.01). The expressions of MYB changed oppositely. During the monocytic differentiation of HL60 cells, miR-16 expression showed a time-dependent increase, but MYB expression gradually decreased. Overexpression of miR-16 in HL60 cells promoted 1,25 D3-induced morphological changes and CD14 expression (p < 0.05). CONCLUSIONS MR-16 facilitated the monocytic differentiation of leukemia HL60 cells by negatively regulating MYB expression.

The Warburg Effect has emerged as a potential drug target because, in some cancer cell lines, it is sufficient to subvert it in order to kill cancer cells. It has also been shown that the Warburg Effect occurs in innate immune cells upon infection. Innate immune cells play critical roles in the tumour microenvironment but the Warburg Effect is not fully understood in monocytes. Furthermore, it is important to understand the impact of infections on key players in the tumour microenvironment because inflammatory conditions often precede carcinogenesis and mutated oncogenes induce inflammation. We investigated the metabolic programme in the acute monocytic leukaemia cell line, THP-1 in the presence and absence of lipopolysaccharide, mimicking bacterial infections. We found that stimulation of THP-1 cells by LPS induces a subset of pro-inflammatory chemokines and enhances the Warburg Effect. Surprisingly, perturbation of the Warburg Effect in these cells does not lead to cell death in contrast to what was observed in non-myeloid cancer cell lines in a previous study. These findings indicate that the Warburg Effect and inflammation are activated by bacterial lipopolysaccharide and may have a profound influence on the microenvironment.

Macrophages are characterized by phenotypical and functional heterogeneity. In different microenvironments, macrophages can polarize into two types: classically activated macrophages (M1) or alternatively activated macrophages (M2). M1 macrophages are a well-known bacteriostatic macrophage, and conversely, M2 macrophages may play an important role in tumor growth and tissue remodeling. M1 macrophages have been reported to have high intracellular iron stores, while M2 macrophages contain lower intracellular iron. It has been well-described that disturbances of iron homeostasis are associated with altered immune function. Thus, it is important to investigate if chronic iron overload is capable of polarizing macrophages. Human monocytic leukemia THP-1 cells were maintained in culture medium that contained 100 μM ferrous sulfate heptahydrate (FeSO4) (I-THP-1) and differentiated into THP-1-derived macrophages (I-TDMs) by induction with phorbol 12-myristate 13-acetate (PMA). We characterized that I-TDMs not only enhanced the surface expression of CD163 and CD206 but also increased arginase and decreased iNOS protein expression. I-TDMs enhanced pSTAT6 expression and decreased pSTAT1 and NF-κB expressions. Furthermore, the gene expression profile of I-TDMs was comparable with M2 macrophages by performing human oligonucleotide DNA microarray analysis. Finally, functional assays demonstrated I-TDMs secreted higher levels of IL-10 but not M1 cytokines. Additionally, the conditional medium of I-TDMs had enhanced migration and increased invasion of A375 melanoma cells which was similar to the characteristics of tumor-associated macrophages. Taken together, we demonstrated that THP-1-derived macrophages polarized to a phenotype of M2-like characteristics when subjected to chronic iron overload.

Notch signaling and nuclear receptor PPARγ are involved in macrophage polarization, but cross talk between them has not been reported in macrophages. In this study, the effect of Notch signaling on PPARγ in IL‐4‐stimulated human macrophages (M(IL‐4)) was investigated using THP‐1‐derived macrophages and human monocyte‐derived macrophages as models. Human M(IL‐4) increased the expression of JAGGED1 and activated Notch signaling. Overexpression of Notch1 intracellular domain (NIC1) increased PPARγ expression, while inhibiting Notch signaling decreased PPARγ levels in M(IL‐4). NIC1 overexpression in THP‐1‐derived macrophages increased PPARγ protein stability by delaying its proteasome‐mediated degradation, but did not affect its mRNA. Phosphorylation of AKT was enhanced in NIC1‐overexpressing cells, and a specific AKT inhibitor reduced the level of PPARγ. NIC1‐overexpressing THP‐1 cells exhibited increased CD36 levels via activation of PPARγ, resulting in enhanced intracellular lipid accumulation. In summary, this study provides evidence linking Notch signaling and PPARγ via AKT in M(IL‐4)., Notch signaling is known to regulate activation of pro‐inflammatory macrophages. Here, we report that IL‐4‐stimulated human macrophages (M(IL‐4)) promptly activate Notch signaling via JAGGED1. This activation is accompanied by increased PPARγ, a key transcription factor for M(IL‐4). Cross talk between Notch signaling and AKT/ERK pathways was observed. Interfering with Notch signaling affects PPARγ level and its biological functions.

Neuroinflammation and microglia-mediated neurotoxicity contribute to the pathogenesis of a broad range of neurodegenerative diseases, therefore, identifying novel compounds that can suppress adverse activation of glia is an important goal. We have previously identified a class of trisubstituted pyrazoles that possess neuroprotective and anti-inflammatory properties. Here, we describe a second generation of pyrazole analogs that were designed to improve their neuroprotective activity toward neurons under inflammatory conditions. Pyrazolyl oxalamide derivatives were designed to explore the effects of steric and electronic factors. Three in vitro assays were performed to evaluate the compounds&rsquo, anti-neurotoxic, neuroprotective, and cytotoxic activity using human THP-1, PC-3, and SH-SY5Y cells. Five compounds significantly reduced the neurotoxic secretions from immune-stimulated microglia-like human THP-1 monocytic cells. One of these compounds was also found to protect SH-SY5Y neuronal cells when they were exposed to cytotoxic THP-1 cell supernatants. While one of the analogs was discarded due to its interference with the cell viability assay, most compounds were innocuous to the cultured cells at the concentrations used (1&ndash, 100 &mu, M). The new compounds reported herein provide a design template for the future development of lead candidates as novel inhibitors of neuroinflammation and neuroprotective drugs.

Inflammasomes are multiprotein complexes that coordinate cellular inflammatory responses and mediate host defense. Following recognition of pathogens and danger signals, inflammasomes assemble and recruit and activate caspase-1, the cysteine protease that cleaves numerous downstream targets, including pro–IL-1β and pro–IL-18 into their biologically active form. In this study, we sought to develop a biosensor that would allow us to monitor the initiation, progression, and resolution of inflammation in living animals. To this end, we inserted a known caspase-1 target sequence into a circularly permuted luciferase construct that becomes bioluminescent upon protease cleavage. This biosensor was activated in response to various inflammatory stimuli in human monocytic cell lines and murine bone marrow–derived macrophages. Next, we generated C57BL/6 transgenic mice constitutively expressing the caspase-1 biosensor. We were able to monitor the spatiotemporal dynamics of caspase-1 activation and onset of inflammation in individual animals in the context of a systemic bacterial infection, colitis, and acute graft-versus-host disease. These data established a model whereby the development and progression of inflammatory responses can be monitored in the context of these and other mouse models of disease.

High-density lipoprotein (HDL) plays a main role in reverse cholesterol transport (RCT), one of the most important functions for preventing atherosclerosis. Recent reports have shown that red blood cells (RBCs) can be associated with RCT, an interaction facilitated by albumin. However, the RCT function of RBCs has not been thoroughly elucidated. In this study, the RCT function of RBCs was assessed using cholesterol efflux capacity (CEC) assays, in which [3H]-labeled cholesterol-loaded human acute monocytic leukemia (THP-1) macrophages were incubated with RBCs as a cholesterol acceptor in the presence or absence of HDL or its main component protein apolipoprotein A-I (apoA-I). The CEC of RBCs was found to be dose dependent, enabling uptake of cholesterol from THP-1 macrophages through apoA-I and HDL, and directly from apoA-I and HDL in medium without the presence THP-1 macrophages. Moreover, RBCs could exchange cholesterol with HDL in a bidirectional manner but could only exchange cholesterol with apoA-I in a single direction. Although albumin promoted the movement of cholesterol, synergistic effects were not observed for both apoA-I and HDL, in contrast to previous findings. These results strongly suggested that RBCs may play important roles in RCT by mediating cholesterol efflux as temporary cholesterol storage.

Previous research has shown that propolis has immunomodulatory activity. Propolis extracts from different geographic origins were assessed for their anti-inflammatory activities by investigating their ability to alter the production of tumour necrosis factor-&alpha, (TNF-&alpha, ) and the cytokines interleukin-1&beta, (IL-1&beta, ), IL-6 and IL-10 in THP-1-derived macrophage cells co-stimulated with lipopolysaccharide (LPS). All the propolis extracts suppressed the TNF-&alpha, and IL-6 LPS-stimulated levels. Similar suppression effects were detected for IL-1&beta, but the release of this cytokine was synergised by propolis samples from Ghana and Indonesia when compared with LPS. Overall, the Cameroonian propolis extract (P-C) was the most active and this was evaluated for its effects on the metabolic profile of unstimulated macrophages or macrophages activated by LPS. The levels of 81 polar metabolites were identified by liquid chromatography (LC) coupled with mass spectrometry (MS) on a ZIC-pHILIC column. LPS altered the energy, amino acid and nucleotide metabolism in THP-1 cells, and interpretation of the metabolic pathways showed that P-C reversed some of the effects of LPS. Overall, the results showed that propolis extracts exert an anti-inflammatory effect by inhibition of pro-inflammatory cytokines and by metabolic reprogramming of LPS activity in macrophage cells, suggesting an immunomodulatory effect.