Untreated THP-1 Cells Research Articles

SPP1/osteopontin: a driver of fibrosis and inflammation in degenerative ascending aortic aneurysm?

Degenerative ascending aortic aneurysm (AscAA) is a silent and potentially fatal disease characterized by excessive vascular inflammation and fibrosis. We aimed to characterize the cellular and molecular signature for the fibrotic type of endothelial mesenchymal transition (EndMT) that has previously been described in degenerative AscAA. Patients undergoing elective open-heart surgery for AscAA and/or aortic valve repair were recruited. Gene expression in the intima-media of the ascending aorta was measured in 22 patients with non-dilated and 24 with dilated aortas, and candidate genes were identified. Protein expression was assessed using immunohistochemistry. Interacting distal gene enhancer regions were identified using targeted chromosome conformation capture (HiCap) in untreated and LPS-treated THP1 cells, and the associated transcription factors were analyzed. Differential expression analysis identified SPP1 (osteopontin) as a key gene in the signature of fibrotic EndMT in patients with degenerative AscAA. The aortic intima-media expression of SPP1 correlated with the expression of inflammatory markers, the level of macrophage infiltration, and the aortic diameter. HiCap analysis, followed by transcription factor binding analysis, identified ETS1 as a potential regulator of SPP1 expression under inflammatory conditions. In conclusion, the present findings suggest that SPP1 may be involved in the development of the degenerative type of AscAA. KEY MESSAGES: In the original manuscript titled "SPP1/osteopontin, a driver of fibrosis and inflammation in degenerative ascending aortic aneurysm?" by David Freiholtz, Otto Bergman, Saliendra Pradhananga, Karin Lång, Flore-Anne Poujade, Carl Granath, Christian Olsson, Anders Franco-Cereceda, Pelin Sahlén, Per Eriksson, and Hanna M Björck, we present novel findings on regulatory factors on osteopontin (SPP1) expression in immune cells involved in degenerative ascending aortic aneurysms (AscAA). The central findings convey: SPP1 is a potential driver of the fibrotic endothelial-to-mesenchymal transition in AscAA. SPP1/osteopontin expression in AscAA is predominately by immune cells. ETS1 is a regulatory transcription factor of SPP1 expression in AscAA immune cells.

11β-Hydroxysteroid dehydrogenase type 1 amplifies inflammation in LPS-induced THP-1 cells.

The role of glucocorticoids as anti-inflammatory and immune-stimulatory drugs has been widely reported. However, the role of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), which catalyzes the conversion of inactive cortisone into active cortisol, in inflammation remains unclear. This study aimed to examine the mechanism of actions of 11β-HSD1 in lipopolysaccharide (LPS)-induced THP-1 cells. The gene expression of 11β-HSD1 and pro-inflammatory cytokines was detected via RT-PCR. The protein expression of IL-1β in cell supernatants was detected via ELISA. Oxidative stress and mitochondrial membrane potential were assessed using a reactive oxygen species (ROS) kit and a mitochondrial membrane potential (MMP) kit, respectively. The expression of Nuclear Factor- Kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) was detected via western blotting. Elevated levels of 11β-HSD1 contributed to the expression of inflammatory cytokines, whereas BVT.2733, a selective 11β-HSD1 inhibitor, ameliorated inflammatory responses, ROS, and mitochondrial damage in LPS-stimulated THP-1 cells. Furthermore, cortisone and cortisol, which are the substrate and product of 11β-HSD1, respectively, showed biphasic responses and induced the expression of pro-inflammatory cytokines at a low concentration in both LPS-stimulated or untreated THP-1 cells. The enhanced inflammation was attenuated by co-treatment with BVT.2733 and the glucocorticoid receptor (GR) antagonist RU486, but not in those treated with the mineralocorticoid receptor (MR) antagonist spironolactone. Overall, the results indicate that 11β-HSD1 amplifies inflammatory responses by activating the NF-κB and MAPK signaling pathways. Inhibition of 11β-HSD1 may serve as a potential therapeutic target against the excessive activation of inflammation.

Expression of the circular RNAs in astaxanthin promotes cholesterol efflux from THP-1 cells based on RNA-seq

BackgroundIt is reported that circular RNAs (circRNAs) play a key role in atherosclerosis (AS). Foam cell formation, which is the main feature of AS, can be significantly inhibited by cholesterol efflux.MethodsWe established a model of astaxanthin (AST) promoting cholesterol efflux from macrophages through oil red O staining, real-time quantitative PCR (qRT-PCR), and western blot and used RNA sequencing to detect the expression of circRNAs in AST-treated and untreated THP-1 cells. Finally, siRNA transfection screened out circRNAs that were significantly differentially expressed. The data analysis was performed by Student’s t test and P < 0.05 was considered statistically significant.ResultsIn the model of AST promoting cholesterol efflux from THP-1 cells, there were a total of 7276 circRNAs differentially expressed, among which the top 25 upregulated and the top 25 downregulated circRNAs were selected based on the log2 (fold change). GO analysis showed that differential expression of circRNAs in biological process (2066/3098; 66.69%), molecular function (543/3098; 17.53%), and cellular component (489/3098; 15.78%). Based on KEGG analysis, RNA transport was the most enriched pathway. Finally, we obtained 3 significantly upregulated circRNAs by siRNA transfection and qRT-PCR.ConclusionsThe 3 differentially expressed circRNAs may play an important role in the process of AST promoting cholesterol efflux and may be used as biomarkers to prevent AS.

Quantitative Structure-Activity Relationship Models for Predicting Inflammatory Potential of Metal Oxide Nanoparticles.

Background:Although substantial concerns about the inflammatory effects of engineered nanomaterial (ENM) have been raised, experimentally assessing toxicity of various ENMs is challenging and time-consuming. Alternatively, quantitative structure–activity relationship (QSAR) models have been employed to assess nanosafety. However, no previous attempt has been made to predict the inflammatory potential of ENMs.Objectives:By employing metal oxide nanoparticles (MeONPs) as a model ENM, we aimed to develop QSAR models for prediction of the inflammatory potential by their physicochemical properties.Methods:We built a comprehensive data set of 30 MeONPs to screen a proinflammatory cytokine interleukin (IL)-1 beta () release in THP-1 cell line. The in vitro hazard ranking was validated in mouse lungs by oropharyngeal instillation of six randomly selected MeONPs. We established QSAR models for prediction of MeONP-induced inflammatory potential via machine learning. The models were further validated against seven new MeONPs. Density functional theory (DFT) computations were exploited to decipher the key mechanisms driving inflammatory responses of MeONPs.Results:Seventeen out of 30 MeONPs induced excess production in THP-1 cells. In vivo disease outcomes were highly relevant to the in vitro data. QSAR models were developed for inflammatory potential, with predictive accuracy (ACC) exceeding 90%. The models were further validated experimentally against seven independent MeONPs (). DFT computations and experimental results further revealed the underlying mechanisms: MeONPs with metal electronegativity lower than 1.55 and positive were more likely to cause lysosomal damage and inflammation.Conclusions: released in THP-1 cells can be an index to rank the inflammatory potential of MeONPs. QSAR models based on were able to predict the inflammatory potential of MeONPs. Our approach overcame the challenge of time- and labor-consuming biological experiments and allowed for computational assessment of MeONP inflammatory potential by characterization of their physicochemical properties. https://doi.org/10.1289/EHP6508

The dialogue between died and viable cells: in vitro and in vivo bystander effects and 1H-NMR-based metabolic profiling of soluble factors

Abstract The bystander effect (BE) is an important biological phenomenon that induces damages in distant and not directly affected by a chemical/physical stress cells. This effect, well known in ionizing radiation treatment, relies on reactive signals released by exposed cells and transmitted via cell–cell interaction or culture medium. In this study, cycloheximide (CHX)-induced apoptotic U937 cells and untreated THP-1 cells were chosen to investigate the chemical-induced BE. The effects of apoptotic U937 cells culture medium, Conditioned Medium (CM), on THP-1 cells were evaluated by morphological and immunohistochemical analysis performed by light microscopy; 1D 1H and 2D J-resolved (JRES) NMR metabolomic analysis has been used to characterize the molecules involved in the BE. In summary, this study indicates that: CM of CHX-treated U937 cells induces a time-dependent induction of toxicity, probably apoptotic cell death, and macrophagic differentiation in THP-1 cells; CM contains different metabolites respect fresh culture medium; CM recruits in vivo activated fibroblasts, endothelial cells, macrophages and mononuclear inflammatory cells in rat calf muscles. These data suggest that CHX exposed cells could cause BE through the release, during the apoptotic process, of soluble factors into the medium that could be exploited in anticancer protocols.

Lysophosphatidylcholine induces expression of genes involved in cholesterol biosynthesis in THP-1 derived macrophages

Lysophosphatidylcholine (LPC), a major component of oxidized low-density lipoprotein, is associated with atherosclerosis, obesity, stroke, and cancer. However, the direction and mechanism of this relationship remains unclear.In this study, we conducted RNA profiling in THP-1 derived macrophages treated with LPC and uncovered a relationship between LPC and the cholesterol biosynthesis pathway. Principal component analysis (PCA) of RNA profiling showed that untreated THP-1 cells and those treated with 10, 20, or 40 µM LPC were distinctly distributed. Functional annotation revealed that LPC affected the expression of genes involved in cytokine–cytokine receptor interaction, TNF signaling, and MAPK signaling. Interestingly, LPC also altered the expression of 11 genes involved in cholesterol synthesis such as those in terpenoid backbone biosynthesis and steroid biosynthesis pathways. This increased gene expression occurred in a dose-dependent manner in response to LPC treatment. Especially, LPC with saturated acyl groups enhanced the expression of these genes compared to LPC with unsaturated acyl groups, and similar results were shown in response to saturated and unsaturated free fatty acids.Our findings demonstrate that LPCs with saturated acyl groups induce the expression of genes involved in cholesterol biosynthesis and may have implications for cholesterol related diseases.

Activation of the monocytic α7 nicotinic acetylcholine receptor modulates oxidative stress and inflammation-associated development of coronary artery spasm via a p38 MAP-kinase signaling-dependent pathway.

Smoking and high-sensitivity C-reactive protein (hs-CRP) are risk factors for coronary artery spasm (CAS), which is characterized by the increased interleukin-6 (IL-6) level and monocyte counts; however, limited data are available regarding the role of cigarette-embedded nicotine in the modulation of monocytic inflammatory activity in CAS. We investigated and elucidated the putative roles and associations of nicotine, monocytic IL-6, α7 nicotinic acetylcholine receptor (α7-nAChR), and CRP in CAS development. We demonstrated that a significantly increased α7-nAChR (p = 0.001) and IL-6 (p = 0.0036) messenger RNA (mRNA) expression in the serum of patients with CAS. Serum hs-CRP levels exhibited a strong positive correlation with the monocytic mRNA expression of α7-nAChR (r = 0.71, p < 0.001) and IL-6 (r = 0.49, p = 0.006). The α7-nAChR and IL-6 expression levels of the CAS group were also positively correlated (r = 0.63, p < 0.001). Compared with the untreated controls, THP-1 cells and patient-derived monocytes treated with different concentrations of CRP displayed significantly increased expression levels of α7-nAChR mRNA and protein (p = 0.0054), in a dose-dependent manner. We also demonstrated that compared with the IL-6 expression elicited by CRP alone (p = 0.0489), the CRP-induced rise in monocytic IL-6 mRNA and protein expression in the presence of nicotine (p = 0.0002), is mediated by α7-nAChR activation and the deregulation of the human p38 mitogen-activated protein kinases (MAPK) signaling pathway. Our data demonstrate that the elevated monocytic IL-6 and α7-nAChR mRNA and protein expression levels are associated with the interaction between nicotine and CRP positively modulates CAS development. Our study suggests the potential role of α7-nAChR mRNA and/or protein expression as a diagnostic biomarker for CAS.

Kinetic Measures of Macrophage Chemotaxis

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

Development of a Pharmacodynamic Assay for XPO1 Occupancy Using Fluorescence Cross Correlation Spectroscopy (FCCS)

Introduction:Selinexor, a Selective Inhibitor of Nuclear Export (SINE) compound, is an anti-cancer drug that is currently being evaluated in Phase I and II clinical trials for the treatment of solid and hematological malignancies (clinicaltrials.gov). XPO1 (exportin 1/CRM1), the target of selinexor, is commonly overexpressed in cancer. XPO1 is the main nuclear export protein with over 200 different protein cargos which include tumor suppressor and growth regulatory proteins. Binding of selinexor to XPO1 inhibits the nuclear export of TSPs and GRPs and induces cell cycle arrest followed by cancer cell apoptosis.Certain cancers are more sensitive to selinexor than others. We hypothesized that part of the inherent differences in sensitivities are attributed to different levels of target inactivation upon selinexor binding. To predict patient response to treatment, we developed a binding assay based on Fluorescence Cross Correlation Spectroscopy (FCCS) to measure XPO1 target occupancy in cancer cells.Methods:Cells were treated in culture for 4 hours with 0-10 µM selinexor. Cells were harvested, lysed, and cleared by ultracentrifugation. In vivo, nude mice were inoculated in both flanks with Z-138 tumor cells. Once tumors reached approximately 250 mm3, mice were administered a single dose of 0, 5, 10, 15, or 20 mg/kg selinexor. Tumors were harvested 6 hours post-dose, lysed, and cleared by ultracentrifugation. Cell or tumor supernatants were incubated with a fluorescently labeled antibody (Ab) against XPO1 and fluorescently labeled Leptomycin B (LMB). To allow binding of labeled Ab and LMB to XPO1, samples were measured by FCCS after 15, 120, and 1200 minutes. FCCS measures signal fluctuations induced by fluorescent molecules diffusing through an illuminated microscopic detection spot. The approach allows simultaneous monitoring of 2 different fluorescently labeled molecules and extracts information on particle concentration, size, binding state, and molecular brightness. Interacting particles are scored by codiffusion. FCCS measurements were performed to quantify the concentration of dually labeled XPO1 molecules in prepared lysates. In the absence of selinexor, complexes of XPO1, the labeled antibody (aXPO1ABATTO488), and the red labeled LMB (LMBDY647) are formed and indicate low target occupancy. XPO1 molecules occupied by selinexor cannot bind to the labeled tracer and decrease the number of dually labeled particles. Accordingly, loss of complex formation is a measure of target occupancy in cells and tumor samples.Results:Comparison of cytotoxicity and XPO1 binding in hematological cell lines treated with increasing concentrations of selinexor in vitro indicated that XPO1 occupancy by selinexor occurred to the same extent regardless of drug sensitivity (Table 1). However, there were two observations that distinguished the selinexor-resistant cell line THP-1 from the other 3 sensitive cell lines (Table 1): 1. Untreated THP-1 cells had the highest XPO1 occupancy of fluorescently labeled LMB compared to lower values in more sensitive cells, and 2. Treatment of THP-1 cells with very low dose selinexor (10 nM) demonstrated lower saturation of XPO1 protein, while treatment of MV-4-11, Z-138, and MM.1S with 10 nM selinexor resulted in marked XPO1 occupancy. These results suggest that high levels of XPO1 proteins might lead to resistance to XPO1 inhibition. We are currently testing whether these two observations are general predictors of resistance in other cell lines. Mouse studies showed that XPO1 target occupancy could be measured in tumors and that there was a dose-dependent effect of selinexor on complex formation with >90% target saturation at 10 mg/kg selinexor (estimated Cmax ~2400 ng/ml).Conclusions: For the first time, the FCCS method permits the direct measurement XPO1 occupancy by selinexor in cells and tumors following treatment. Here, we demonstrate XPO1 occupancy saturation at 10 mg/kg (30 mg/m2) in mice, a dose active against xenografts, and consistent with doses of selinexor that show anti-cancer activity in patients with heavily pretreated hematologic and solid tumors.Table 1Cytotoxicity and FCCS values for cell lines.Cell LineMTT IC50 (nM)FCCS IC50 (nM)FCCS Complex without selinexor (nM)FCCS Complex with 10 nM selinexor (nM)THP-1106035.53.953.19MV-4-112056.22.551.9Z-1384024.00.720.66MM.1S2020.42.251.64 DisclosuresCrochiere:Karyopharm: Employment. Hannus:Intana: Employment. Hansen:Intana: Employment. Becker:Intana: Employment. Ellis:Karyopharm Therapeutics Inc: Employment. Lee:Karyopharm Therapeutics Inc: Employment. Landesman:Karyopharm: Employment.

Human trophoblasts cells modulate the occurrence of apoptosis in monocytes infected by Toxoplasma gondii

s / Placenta 36 (2015) 469e521 520 Methods: For this purpose, HTR-8/SVneo cells were treated or not with the supernatant from uninfected or infectedmacrophages and then infected or not by T. gondii. Cytokine secretion and soluble FasL were analyzed by ELISA, apoptosis index and Fas/CD95 expression were determined by flow cytometry and intracellular parasite proliferation was analyzed by enzymatic assay. Results: IL-6 secretion by HTR-8/SVneo cells was synergistically increased with both stimuli, supernatant from macrophages and T. gondii infection. The apoptosis index of infected HTR-8/SVneo cells was decreased in the presence or absence of supernatant from infected macrophages. On the other hand, the apoptosis of infected HTR-8/SVneo cells increased when these cells were treated with supernatant from uninfected macrophages. Also, a low expression of Fas/CD95 and a high soluble FasL release were observed in these conditions. Finally, we did not verify any change in the proliferation of T. gondii. However, there is down-modulation of apoptosis in HTR-8/SVneo cells by the parasite, which probably relates to favoring its establishment in the host cell, while the functional activities of macrophages toward restoring these cell death rates, keep gestational events of invasion and placentation appropriate. Conclusions: All together, these results contribute to better understanding the mechanisms of cellular communication between the extravillous trophoblast cells and macrophages, and demonstrate that infection with T. gondii can interfere with this interaction through the modulation of cell death by apoptosis. PB.47. HUMAN TROPHOBLASTS CELLS MODULATE THE OCCURRENCE OF APOPTOSIS IN MONOCYTES INFECTED BY TOXOPLASMA GONDII A.S. Castro , F.C. Oliveira , P.S.G. Gois , C.M.O.S. Alves , M.B. Angeloni , P.M. Guirelli , R.J. Silva , P.S. Franco , A.O. Gomes , B.F. Barbosa , T.W.P. Mineo , E.A.V. Ferro . 1 Institute of Biomedical Science, Histology and Embryology Department, Federal University Uberlandia, Uberlandia, Brazil; 2 Institute of Biomedical Science, Pharmacology Department, Federal University Uberlandia, Uberlandia, Brazil; 3 Institute of Biomedical Science, Immunoparasitology Department, Federal University Uberlandia, Uberlandia, Brazil Human trophoblast cells modulate the occurrence of apoptosis in monocytes infected by Toxoplasma gondii Objectives: a) to evaluate the role of trophoblast cells (BeWo) in monocyte (THP-1) activity, specifically apoptosis, in the presence or absence of T. gondii infection; b) to investigate whether BeWo cells alter the expression of death receptor (CD95) in THP-1 cells; c) to investigate FasL release by BeWo and THP-1 cells as well as in THP-1 cells stimulated with supernatant from BeWo cells. Methods: THP-1 cells were stimulated with supernatants of BeWo cells previously infected or not with T. gondii, and then infected with parasites. Supernatant of both cells was collected and analyzed for FasL secretion by ELISA. Furthermore, the occurrence of apoptosis in THP-1 cells and the expression of death receptor (CD95) in these cells were evaluated by flow cytometry. Results: The apoptosis index in THP-1 cells stimulated with supernatants of BeWo cells was higher than in untreated THP-1 cells. In the group of infected THP-1 cells, the apoptosis index decreased at all conditions when compared to the group of uninfected THP-1 cells. However, the expression of death receptor (CD95) was higher in the group of infected THP-1 cells and the stimulus with supernatants of BeWo cells induced a higher expression of this receptor in THP-1 cells. Finally, the secretion of FasL was higher in the group of infected THP-1 cells and supernatant of BeWo cells induced the reduction of FasL secretionwhen compared to controls in both groups. Conclusions: Trophoblast cells and T. gondii alter the occurrence of apoptosis, expression of death receptor (CD95) and secretion of FasL in monocytes. These data can be associated with an immune response established by trophoblast cells and monocytes to protect the fetus against infection, although these findings can also represent a potential survival mechanism of the parasite. PB.48. CHLORPYRIFOS EXPOSURE INDUCES TROPHOBLAST BARRIER AND STROMA ALTERATIONS IN HUMAN CHORIONIC VILLOUS EXPLANTS M.E. Ridano , A.C. Racca , J.B. Flores-Martin , E. Bevilacqua , S. GentiRaimondi , R.E. Fretes , G.M. Panzetta-Dutari . CIBICI-CONICET, Departamento de Bioquimica Clinica, Facultad de Ciencias Quimicas, Universidad Nacional C ordoba, Argentina; 2 Institute of Biomedical Sciences, University of S~ ao Paulo, S~ ao Paulo, Brazil; Departmento de Histologia y Embriologia, Facultad de Medicina, Universidad Nacional

Heat-stable molecule derived from Streptococcus cristatus induces APOBEC3 expression and inhibits HIV-1 replication.

Although most human immunodeficiency virus type 1 (HIV-1) cases worldwide are transmitted through mucosal surfaces, transmission through the oral mucosal surface is a rare event. More than 700 bacterial species have been detected in the oral cavity. Despite great efforts to discover oral inhibitors of HIV, little information is available concerning the anti-HIV activity of oral bacterial components. Here we show that a molecule from an oral commensal bacterium, Streptococcus cristatus CC5A can induce expression of APOBEC3G (A3G) and APOBEC3F (A3F) and inhibit HIV-1 replication in THP-1 cells. We show by qRT-PCR that expression levels of A3G and A3F increase in a dose-dependent manner in the presence of a CC5A extract, as does A3G protein levels by Western blot assay. In addition, when the human monocytic cell line THP-1 was treated with CC5A extract, the replication of HIV-1 IIIB was significantly suppressed compared with IIIB replication in untreated THP-1 cells. Knock down of A3G expression in THP-1 cells compromised the ability of CC5A to inhibit HIV-1 IIIB infectivity. Furthermore, SupT1 cells infected with virus produced from CC5A extract-treated THP-1 cells replicated virus with a higher G to A hypermutation rate (a known consequence of A3G activity) than virus used from untreated THP-1 cells. This suggests that S. cristatus CC5A contains a molecule that induces A3G/F expression and thereby inhibits HIV replication. These findings might lead to the discovery of a novel anti-HIV/AIDS therapeutic.

HIV-1 incorporation of host-cell–derived glycosphingolipid GM3 allows for capture by mature dendritic cells

The interaction between HIV and dendritic cells (DCs) is an important early event in HIV-1 pathogenesis that leads to efficient viral dissemination. Here we demonstrate a HIV gp120-independent DC capture mechanism that uses virion-incorporated host-derived gangliosides with terminal α2-3-linked sialic acid linkages. Using exogenously enriched virus and artificial liposome particles, we demonstrate that both α2-3 gangliosides GM1 and GM3 are capable of mediating this interaction when present in the particle at high levels. In the absence of overexpression, GM3 is the primary ligand responsible for this capture mechanism, because siRNA depletion of GM3 but not GM1 from the producer cell and hence virions, resulted in a dramatic decrease in DC capture. Furthermore, HIV-1 capture by DCs was competitively inhibited by targeting virion-associated GM3, but was unchanged by targeting GM1. Finally, virions were derived from monocytoid THP-1 cells that constitutively display low levels of GM1 and GM3, or from THP-1 cells induced to express high surface levels of GM1 and GM3 upon stimulation with the TLR2/1 ligand Pam3CSK4. Compared with untreated THP-1 cells, virus produced from Pam3CSK4-stimulated THP-1 cells incorporated higher levels of GM3, but not GM1, and showed enhanced DC capture and trans-infection. Our results identify a unique HIV-1 DC attachment mechanism that is dependent on a host-cell-derived ligand, GM3, and is a unique example of pathogen mimicry of host-cell recognition pathways that drive virus capture and dissemination in vivo.

Uncoupling between CD1d upregulation induced by retinoic acid and conduritol-B-epoxide and iNKT cell responsiveness

Gaucher disease (GD) is associated with upregulation of CD1d and MHC-class II expression by monocytes. While the physiological impact of CD1d upregulation remains uncertain, it has been proposed that MHC-class II upregulation is associated with inflammation. Hereby, we show that the decrease in MHC-class II expression seen in GD patients under therapy correlates positively with chitotriosidase activity, a marker of inflamed macrophages. We also show that retinoic acid (RA) and the β-glucocerebrosidase inhibitor conduritol-B-epoxide (CBE) lead to upregulation of CD1d expression by THP-1 cells, which correlated with an increase in mRNA expression. In vitro co-culture experiments showed that RA treated THP-1 cells were more stimulatory for CD4 + than for CD8 + T cells, as determined by CFSE loss, in comparison to untreated THP-1 cells. Interestingly, even though addition of exogenous isoglobotrihexosylceramide (iGb3), a physiological CD1d ligand, augmented the percentage of dividing CD4 + T cells, we could not detect a significant expansion of CD4 +Vα24 + invariant Natural Killer T (iNKT) cells. In contrast, addition of α-galactosylceramide (α-GC) induced expansion of Vα24 + iNKT cells as determined by using α-GC-loaded human CD1d dimers. These results strengthen the existence of a cross-talk between monocyte lipid accumulation, inflammation and changes in cell surface CD1d and MHC-class II in monocytes, which may result in inappropriate recognition events by immune cells and perpetuate chronic inflammation.

Down-regulation of HOXA4, HOXA7, HOXA10, HOXA11 and MEIS1 during monocyte-macrophage differentiation in THP-1 cells

The translocation t(9;11)(p22;q23) generates the MLL-AF9 oncogene and is commonly associated with monocytic acute myeloid leukemia (AML-M5; FAB-classification). For the oncogenicity of MLL-AF9, the (over)expression of several other genes, including selected HOXA cluster genes as well as MEIS1 (a HOX cofactor), is required. We previously showed that the down-regulation of MLL-AF9 expression is not obligatory for monocyte-macrophage maturation in AML-M5 cells carrying t(9;11)(p22;q23). In this study, we analyzed the expression patterns of HOXA4, 5, 6, 7, 9, 10 and 11 (defined as 'HOXA-code' genes) and MEIS1 by semiquantitative RT-PCR during the monocyte-macrophage differentiation induced by phorbol 12-myristate 13-acetate (PMA) in THP-1 cells carrying t(9;11)(p22;q23) and expressing MLL-AF9. The analyses were performed in THP-1 cells expressing MLL-AF9 even after PMA treatment. The results showed that all the analyzed genes were expressed in untreated THP-1 cells. After the induction of differentiation, we observed a down-regulation of HOXA4, 7, 10, 11 and MEIS1, an up-regulation of HOXA6, and no significant variation in the expression of HOXA5 and 9. These data indicate that the expression of most HOXA-code genes, as well as MEIS1, could be implicated in the differentiation blockage observed in MLL-AF9-related leukemias.

Human chorionic villous macrophages as a fetal biological shield from maternal chorionic gonadotropin

In addition to its most well characterized biological role in the rescue and maintenance of corpus luteum function, human chorionic gonadotropin (hCG) also stimulates the onset of fetal gonadal steroidogenesis. However, excess hCG is teratogenic to fetal gonadal tissues, and therefore hCG must be tightly regulated. Although there is an anatomical barrier between the fetal vessels and maternal blood, other mechanisms may regulate hCG levels. In the present study, we investigated whether human chorionic villous macrophages degraded maternal hCG. Isolated human macrophages incorporated and degraded hCG in a time-dependent manner. Human placental villous macrophages and phorbol myristate acetate (PMA)-treated THP-1 cells expressed the gene encoding an exon 9-deleted form of the luteinizing hormone/chorionic gonadotropin (LH/CG) receptor; expression of the full-length receptor was not determined. While both PMA-treated or untreated THP-1 cells could uptake hCG into their cytoplasms, hCG degradation and excretion of its byproducts only progressed in PMA-treated THP-1 cells. In conclusion, hCG internalization and degradation are different processes in macrophages that protect fetal gonadogenesis from excess hCG. The exon 9-deleted LH/CG receptor, but not the full-length receptor, is involved in the degradation of cytoplasmic hCG by organ-specific, dominant-negative interactions.

Activation of the Inflammatory Cytokine Genes by Intracellular Galectin-9 in Human Monocytes.

Galectins are a family member of animal lectins that function both extracellularly (by interacting with cell surface and extracellular matrix glycoproteins and glycolipids) and intracellularly (by interacting with cytoplasmic and nuclear proteins) to modulate signaling pathways. To date, 15 galectins have been cloned in mammals. The members can be classified into 3 subtypes according to their structure. The proto type and chimera type galectins have a single CRD. The tandem-repeat type galectins have two CRDs. In this regard, galectin-9 is a tandem repeat type galectin, which has been identified as a tumor antigen of unknown functions in patients with Hodgkin lymphoma. Galectin-9 has recently demonstrated to induce chemotaxis of eosinophils and apoptosis in a variety of cells, resulting in immunomodulation, inflammation and anti-metastatic potential of tumor cells. However, the role of galectin-9 in the inflammatory responses still remains unclear. In the present study, we investigated whether intracellular galectin-9 contributes to activation of the inflammatory cytokine genes such as IL-1α, IL-1β and IFNγin human monocytes. Protein expression of galectin-9 in human THP-1 monocytic cells was examined in western blot with anti-galectin-9 Ab and immunostaining studies using a confocal microscopy. A galectin-9 expression vector pBKCMV3-Galectin-9 was transiently cotransfected into THP-1 cells along with luciferase reporter plasmids for IL-1α, IL-1β and IFNγgene promoters. Galectin-9 was detected in the cytoplasm of untreated THP-1 cells, and the expression levels of galectin-9 protein was significantly enhanced in THP-1 cells treated with lipopolysaccharide (LPS). In transient transfection studies, overexpression of galectin-9 dose-dependently activated IL-1α, IL-1β and IFNγgene promoters in THP-1 cells, showing that intracellular galectin-9 has capacity to induce the inflammatory cytokine genes in monocytes. This argument was further supported by our reverse transcription-polymerase chain reaction (RT-PCR) data that galectin-9 overexpression enhanced mRNA levels of IL-1α and β in THP-1 cells. When luciferase reporter plasmids for transcription factors including NF-IL6, NF-κB and AP-1 were transiently cotransfected into THP-1 cells along with pBKCMV3-Galectin-9, NF-IL6 and AP-1 activities were induced by galectin-9 overexpression. Our coimmunoprecipitation study, using anti-galectin-9 Ab and anti-NF-IL6 Ab revealed that intracellular galectin-9 was physically associated with NF-IL6. On the other hand, galectin-9 failed to induce NF-κB activity. Additionally, in contrast to the data obtained from transient transfection studies using pBKCMV3-Galectin-9, no significant induction of IL-1α promoter activity was observed by adding galectin-9 protein to THP-1 cell culture medium, indicating that extracellular galectin-9 dose not affect the inflammatory cytokine gene regulation. Our results strongly suggested that intracellular galectin-9 has the capability to transactivate the inflammatory cytokine genes through the physical interaction with leucin zipper transcription factors such as NF-IL6 in monocytes.

Binding of Caspase-3 Prodomain to Heat Shock Protein 27 Regulates Monocyte Apoptosis by Inhibiting Caspase-3 Proteolytic Activation

Caspase-3 is an essential executioner of apoptosis responsible for regulating many important cellular processes, among them the number of circulating monocytes, central players in the innate immune response. The activation of caspase-3 requires its processing from an inactive precursor. Here we show that the small heat shock protein 27 (Hsp27) associates with caspase-3 and protein-protein interaction experiments in vivo and with purified proteins demonstrate a direct interaction between Hsp27 and the amino-terminal prodomain of caspase-3. Using an in vitro caspase-3 activation assay, our results further establish that the interaction of Hsp27 with the caspase-3 prodomain inhibits the second proteolytic cleavage necessary for caspase-3 activation, revealing a novel mechanism for the regulation of this effector caspase. Hsp27 expression in monocytes is constitutive. Consistent with a central role of Hsp27 in blocking caspase-3 activation, Hsp27 down-regulation by double-stranded RNA interference induces apoptosis of macrophages, whereas Hsp27 overexpression increases the life span of monocytes by inhibiting apoptosis. Highlighting the importance of cell partitioning in the regulation of apoptosis, immunofluorescence, and subcellular fractionation studies revealed that whereas both caspase-3 and Hsp27 are cytoplasmic in fresh monocytes (i.e. not undergoing apoptosis), Hsp27 moves to the nucleus during apoptosis, a relocalization that can be blocked by promoting the differentiation of monocytes to macrophages or by inhibiting cell death. These results reveal a novel mechanism of caspase-3 regulation and underscore a novel and fundamental role of Hsp27 in the regulation of monocyte life span.

Effects of the β-Amyloid and Carboxyl-terminal Fragment of Alzheimer's Amyloid Precursor Protein on the Production of the Tumor Necrosis Factor-α and Matrix Metalloproteinase-9 by Human Monocytic THP-1

To explore the direct role of beta-amyloid (Abeta) and carboxyl-terminal fragments of amyloid precursor protein in the inflammatory processes possibly linked to neurodegeneration associated with Alzheimer's disease, the effects of the 105-amino acid carboxyl-terminal fragment (CT(105)) of amyloid precursor protein on the production of tumor necrosis factor-alpha (TNF-alpha) and matrix metalloproteinase-9 (MMP-9) were examined in a human monocytic THP-1 cell line and compared with that of Abeta. CT(105) elicited a marked increase in TNF-alpha and MMP-9 production in the presence of interferon-gamma in a dose- and time-dependent manner. Similar patterns were obtained with Abeta despite its low magnitude of induction. Autocrine TNF-alpha is likely to be a main mediator of the induction of MMP-9 because the neutralizing antibody to TNF-alpha inhibits MMP-9 production. Genistein, a specific inhibitor of tyrosine kinase, dramatically diminished both TNF-alpha secretion and subsequent MMP-9 release in response to CT(105) or Abeta. Furthermore, PD98059 and SB202190, specific inhibitors of ERK or p38 MAPK respectively, efficiently suppressed CT(105)-induced effects whereas only PD98059 was effective at reducing Abeta-induced effects. Our results suggest that CT(105) in combination with interferon-gamma might serve as a more potent activator than Abeta in triggering inflammatory processes and that both tyrosine kinase and MAPK signaling pathways may represent potential therapeutic targets for the control of Alzheimer's disease progression.

Transcriptional control of adrenomedullin induction by phorbol ester in human monocytic leukemia cells.

Adrenomedullin is a potent vasodilator peptide that was originally identified from human pheochromocytoma. In this study, we investigated the induction of adrenomedullin gene expression in THP-1 acute monocytic leukemia cells during differentiation into macrophage-like cells by 12-O-tetradecanoylphorbol-13-acetate (TPA), and identified a cis-regulatory region of the human adrenomedullin gene responsible for TPA-induced adrenomedullin expression. Upon treatment with TPA (100 ng x mL(-1)) for 24 h, immunoreactive adrenomedullin concentrations in the culture medium and adrenomedullin mRNA levels were increased more than 10-fold, concomitant with the differentiation of THP-1 cells into macrophage-like cells. Actinomycin D abolished the TPA-induced adrenomedullin expression, indicating that the induction of ADM gene expression by TPA was regulated at the transcriptional level. Transient transfection assay revealed that a cis-acting region (positions -70 to -30) of human adrenomedullin gene was necessary for TPA-induced reporter gene expression. This region contains multiple copies of activator protein 2 (AP-2) binding sites, which are bound by purified AP-2 protein, as judged by electrophoretic mobility shift assay. The binding activity to this region was undetectable in nuclear extracts prepared from untreated THP-1 cells, but was increased in extracts prepared from TPA-treated cells. The protein binding was abolished by unlabeled oligonucleotides containing the AP-2 consensus sequence. These results indicate that the region (-70 to -30) of the human ADM gene containing multiple AP-2 binding sites is responsible for TPA-induced adrenomedullin expression in THP-1 cells.

Antigen activation of THP-1 human monocytic cells after stimulation with lipopolysaccharide from oral microorganisms and granulocyte-macrophage colony-stimulating factor.

A human THP-1 monocyte cell line culture system has been utilized to evaluate the morphological changes in THP-1 cells and to measure expression of activation antigens (CD-11b, CD-11c, CD-14, CD-35, CD-68, CD-71 and HLA-DR) as evidence of maturation of THP-1 cells in response to stimulation by lipopolysaccharide (LPS) from the oral microorganisms, Fusobacterium nucleatum and Porphyromonas gingivalis, and granulocyte-macrophage colony-stimulating factor. THP-1 cells were stimulated with LPS (1 microgram/ml) of P. gingivalis or F. nucleatum for different time periods (1, 2, 4 and 7 d). Detection of different activation antigens on THP-1 cells was performed by indirect immunohistochemical staining followed by light microscopy. Confirmational studies were performed in parallel using indirect immunofluorescence and immunogold electron microscopy for detection of the corresponding activation antigens. Expression of different activation antigens by resting THP-1 cells revealed HLA-DR to be on 3% of the cells; CD-11b, 9%; CD-11c, 8%; CD-14, 22%; CD-35, 9% and CD-68, 7%. The CD-71 activation antigen was not expressed in untreated THP-1 cells. LPS stimulation increased expression of all activation antigens. A significant (p < 0.05) increase in expression of CD-11b, CD-11c, CD-14, CD-35, CD-68 and CD-71 was observed when GM-CSF (50 IU/ml) was supplemented during the treatment of THP-1 cells with LPS of F. nucleatum or P. gingivalis. Activation and differentiation of THP-1 cells by LPS from oral microorganisms in the presence of GM-CSF supports a role for human macrophages in acute and chronic periodontal diseases and may explain the clinically observable periodontal exacerbations in some patients after GM-CSF therapy.