Iron Must Be in Haemin to Act as a Pro-Inflammatory Stimulus in Cultured Human THP-1 Monocytes.

Study on Sucrosomial® Iron Endocytosis-Mediated Uptake and Enterocytes Release

Terbinafine stimulates the pro-inflammatory responses in human monocytic THP-1 cells through an ERK signaling pathway

The nuclear factor (NF)-kappaB/inhibitory (I)kappaBalpha pathway is one of the most important intracellular signal transduction pathways during inflammation which is induced by a variety of major early response cytokines. Recent studies suggest that volatile anesthetics interfere with inflammatory cytokine production through inhibition of intracellular signal transduction pathways. We, therefore, aimed to investigate the effects of the volatile anesthetics sevoflurane and isoflurane on NF-kappaB/IkappaBalpha-dependent intracellular signal transduction in human monocytic THP-1 cells induced by tumor necrosis factor-alpha (TNF-alpha) and production of interleukin-8 (IL-8) and downstream heme oxygenase-1 (HO-1). THP-1 cells, a human monocytic cell line, were used in an in vitro model which enables the exposure to volatile anesthetics. Using this model, THP-1 cells were subjected to sevoflurane or isoflurane exposure (1 MAC each) and were stimulated with TNF-alpha (50 or 100 ng/ml). Compared to untreated cells, expression of intracellular HO-1-protein and release of IL-8 into cell culture supernatants and corresponding mRNA expression were attenuated in THP-1 cells exposed to sevoflurane and isoflurane, respectively. Moreover, translocation of NF-kappaB and degradation of IkappaBalpha were markedly reduced by both anesthetics. Notably, under unstimulated conditions, exposure to sevoflurane induced a sustained upregulation of the IkappaBalpha content in THP-1 cells. We demonstrated inhibition of TNF-alpha-induced gene expression and release of IL-8 and HO-1 in human monocytic THP-1 cells exposed to both volatile anesthetics. This was associated with an upregulated intracellular IkappaBalpha content followed by decreased NF-kappaB translocation. This was more sustained during exposure to sevoflurane and may provide an additional intracellular mechanism for the anti-inflammatory effects associated with sevoflurane administration.

After haemin treatment intracellular non-haem iron increases prior to haem oxygenase-1 induction: A study in human monocytic cell line THP-1

Pro-inflammatory cytokines and anti-inflammatory cytokines are important mediators that regulate the inflammatory response in inflammation-related diseases. The aim of this study is to evaluate different New Zealand (NZ)-grown ginseng fractions on the productions of pro-inflammatory and anti-inflammatory cytokines in human monocytic THP-1 cells. Four NZ-grown ginseng fractions, including total ginseng extract (TGE), non-ginsenoside fraction extract (NGE), high-polar ginsenoside fraction extract (HPG), and less-polar ginsenoside fraction extract (LPG), were prepared and the ginsenoside compositions of extracts were analyzed by HPLC using 19 ginsenoside reference standards. The THP-1 cells were pre-treated with different concentrations of TGE, NGE, HPG, and LPG, and were then stimulated with lipopolysaccharide (LPS). The levels of pro-inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), and anti-inflammatory cytokines, such as interleukin-10 (IL-10), and transforming growth factor beta-1 (TGF-β1), were determined by enzyme-linked immunosorbent assay (ELISA). TGE at 400 µg/mL significantly inhibited LPS-induced TNF-α and IL-6 productions. NGE did not show any effects on inflammatory secretion except inhibited IL-6 production at a high dose. Furthermore, LPG displayed a stronger effect than HPG on inhibiting pro-inflammatory cytokine (TNF-α, IL-1β, and IL-6) productions. Particularly, 100 µg/mL LPG not only significantly inhibited the production of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6, but also remarkably enhanced the production of anti-inflammatory cytokine IL-10. NZ-grown ginseng exhibited anti-inflammatory effects in vitro, which is mainly attributed to ginsenoside fractions (particularly less-polar ginsenosides) rather than non-saponin fractions.

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.

Infection of macrophages with mycobacteria has been shown to inhibit the macrophage response to IFN-gamma. In the current study, we examined the effect of Mycobacteria avium, Mycobacteria tuberculosis, and TLR2 stimulation on IFN-gamma-induced gene expression in human PMA-differentiated THP-1 monocytic cells. Mycobacterial infection inhibited IFN-gamma-induced expression of HLA-DRalpha and HLA-DRbeta mRNA and partially inhibited CIITA expression but did not affect expression of IFN regulatory factor-1 mRNA. To determine whether inhibition of histone deacetylase (HDAC) activity could rescue HLA-DR gene expression, butyric acid and MS-275, inhibitors of HDAC activity, were added at the time of M. avium or M. tuberculosis infection or TLR2 stimulation. HDAC inhibition restored the ability of these cells to express HLA-DRalpha and HLA-DRbeta mRNA in response to IFN-gamma. Histone acetylation induced by IFN-gamma at the HLA-DRalpha promoter was repressed upon mycobacteria infection or TLR2 stimulation. HDAC gene expression was not affected by mycobacterial infection. However, mycobacterial infection or TLR2 stimulation up-regulated expression of mammalian Sin3A, a corepressor that is required for MHC class II repression by HDAC. Furthermore, we show that the mammalian Sin3A corepressor is associated with the HLA-DRalpha promoter in M. avium-infected THP-1 cells stimulated with IFN-gamma. Thus, mycobacterial infection of human THP-1 cells specifically inhibits HLA-DR gene expression by a novel pathway that involves HDAC complex formation at the HLA-DR promoter, resulting in histone deacetylation and gene silencing.

Signal transduction of phorbol 12-myristate 13-acetate (PMA)-induced growth inhibition of human monocytic leukemia THP-1 cells is reactive oxygen dependent

Interleukin-21 (IL-21) is a type I cytokine produced by activated T cells that promotes cytokine production in monocytes. Monocytes are activated by Toll-like receptors (TLRs) to produce pro-inflammatory mediators. However, little is known about the regulatory effect of IL-21 on TLR-mediated inflammation in human monocytes. This study investigated the potential association between IL-21 and TLR2/4-mediated inflammation in human monocytic THP-1 cells. First, the expression of the IL-21 receptor (IL-21R) in human monocytic THP-1 cells was examined by flow cytometry. Then, THP-1 cells were treated with either the TLR2 ligand peptidoglycan (PGN) or the TLR4 ligand lipopolysaccharide (LPS) with or without IL-21. Then, the production of several cytokines (IL-6, IL-8, TNF-α, IFN-γ and IL-10), expression of TLR2/4, and activation of the downstream signaling pathways of mitogen-activated protein kinase (MAPK), Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3), phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt), and nuclear factor-kappa B (NF-κB) were determined. We found that IL-21R was highly expressed in human monocytic THP-1 cells and that IL-21 induced TLR2 and TLR4 expression and further enhanced PGN/LPS-mediated TLR2/4 expression. In addition, IL-21 also upregulated the expression of IL-6, IL-8, TNF-α, IFN-γ and IL-10 and enhanced TLR2/4-mediated cytokine production in THP-1 cells via phosphorylation of the STAT3, Akt and p38 MAPK signalling pathways. Our study suggests, for the first time that IL-21 enhances TLR2/4-mediated cytokine production in human monocytic THP-1 cells by activating the STAT3, PI3K/Akt and p38 MAPK signalling pathways.

The ability of Coxiella burnetii to modulate host cell death may be a critical factor in disease development. In this study, human monocytic THP-1 cells were used to examine the ability of C. burnetii Nine Mile phase II (NMII) to modulate apoptotic signaling. Typical apoptotic cell morphological changes and DNA fragmentation were detected in NMII infected cells at an early stage of infection. FACS analysis using Annexin-V-PI double staining showed the induction of a significant number of apoptotic cells at an early stage of NMII infection. Double staining of apoptotic cell DNA and intracellular C. burnetii indicates that NMII infected cells undergoing apoptosis. Interestingly, caspase-3 was not cleaved in NMII infected cells and the caspase-inhibitor Z-VAD-fmk did not prevent NMII induced apoptosis. Surprisingly, the caspase-3 downstream substrate PARP was cleaved in NMII infected cells. These results suggest that NMII induces apoptosis during an early stage of infection through a caspase-independent pathway in THP-1 cells. In addition, NMII-infected monocytes were unable to prevent exogenous staurosporine-induced apoptotic death. Western blot analysis indicated that NMII infection induced the translocation of AIF from mitochondria into the nucleus. Cytochrome c release and cytosol-to-mitochondrial translocation of the pore-forming protein Bax in NMII infected cells occurred at 24 h post infection. These data suggest that NMII infection induced caspase-independent apoptosis through a mechanism involving cytochrome c release, cytosol-to-mitochondrial translocation of Bax and nuclear translocation of AIF in THP-1 monocytes. Furthermore, NMII infection increased TNF-α production and neutralization of TNF-α in NMII infected cells partially blocked PARP cleavage, suggesting TNF-α may play a role in the upstream signaling involved in NMII induced apoptosis. Antibiotic inhibition of C. burnetii RNA synthesis blocked NMII infection-induced PARP activation. These results suggest that both intracellular C. burnetii replication and secreted TNF-α contribute to NMII infection-triggered apoptosis during an early stage of infection.

Microvesicle-mediated RNA Molecule Delivery System Using Monocytes/Macrophages

Objective To observe the effect of simulated microgravity on lipopolysaccharide (LPS)-induced inflammatory cytokines release in human monocytic leukemia THP-1 cells. Methods The human′s THP-1 cells were chosen as the research objects and divided into control group and simulated weightlessness group. After being resuscitated and subcultured, the cells in simulated weightlessness group were treated by clinostat with the speed of 30 r/min and cultured in the 37℃ incubator. The control group was set up synchronously with the experimental conditions that were consistent with simulated weightlessness group but without clinostat treatment. Six bottles of cells for each group had been tested for 3 times. When the cells in simulated weightlessness group were cultured in the clinostat for 48 h, 3 bottles of cell for each group were centrifuged for 5 min with the speed of 1 000 r/min. Then the LPS cell was added to stimulate cells with the concentration of 1 μg/ml and the cultivation lasted for 8 h. The cells and the cultured medium were collected by centrifugation. The other 3 bottles of control group and simulated weightlessness group were added equivalent volume cultured medium with LPS. The cells and the cultured medium were collected by centrifugation 8 h later. The experiment was repeated for 3 times. The mRNA expression and the changes of the concentration of TNF-α, IL-6, IL-1β gathered under 4 experimental conditions were compared. Results ①Compared with those in the control group, the mRNA expressions of TNF-α, IL-6 and IL-1β were increased significantly in LPS group and simulated weightlessness + LPS group (q=11.63-50.24, P<0.05); mRNA expressions of TNF-α and IL-1β were increased significantly in simulated weightlessness group (q=5.56-3.44, P<0.05). Compared with LPS group, the mRNA expressions of TNF-α and IL-6, IL-1β were increased significantly in simulated weightlessness + LPS group (q=9.08-26.50, P<0.01). ②Compared with those in control group, the concentration of TNF-α, IL-6, IL-1β were increased significantly in LPS group and simulated weightlessness group (q=3.02-32.52, P<0.05); the concentrations of TNF-α, IL-6 and IL-1β were increased significantly in simulated weightlessness + LPS group (q=40.02-65.31, P<0.01). Compared with those in LPS group, the concentrations of TNF-α and IL-6, IL-1β were increased significantly in simulated weightlessness + LPS group (q=17.59-32.79, P<0.01). Conclusions Simulated weightlessness can significantly increase the secretion of inflammatory cytokines and aggravate the LPS-induced inflammatory reaction in THP-1 cells. Key words: Weightlessness simulated; Lipopolysaccharides; Gene expression; Cytokines

GTP cyclohydrolase I feedback regulatory protein (GFRP) is a 9.7-kDa protein regulating GTP cyclohydrolase I activity in dependence of tetrahydrobiopterin and phenylalanine concentrations, thus enabling stimulation of tetrahydrobiopterin biosynthesis by phenylalanine to ensure its efficient metabolism by phenylalanine hydroxylase. Here, we were interested in regulation of GFRP expression by proinflammatory cytokines and stimuli, which are known to induce GTP cyclohydrolase I expression. Recombinant human GFRP stimulated recombinant human GTP cyclohydrolase I in the presence of phenylalanine and mediated feedback inhibition by tetrahydrobiopterin. Levels of GFRP mRNA in human myelomonocytoma (THP-1) cells remained unaltered by treatment of cells with interferon-gamma or interleukin-1beta, but were significantly down-regulated by bacterial lipopolysaccharide (LPS, 1 microg/ml), without or with cotreatment by interferon-gamma, which strongly up-regulated GTP cyclohydrolase I expression and activity. GFRP expression was also suppressed in human umbilical vein endothelial cells treated with 1 microg/ml LPS, as well as in rat tissues 7 h post intraperitoneal injection of 10 mg/kg LPS. THP-1 cells stimulated with interferon-gamma alone showed increased pteridine synthesis by addition of phenylalanine to the culture medium. Cells stimulated with interferon-gamma plus LPS, in contrast, showed phenylalanine-independent pteridine synthesis. These results demonstrate that LPS down-regulates expression of GFRP, thus rendering pteridine synthesis independent of metabolic control by phenylalanine.

Lipoic acid ameliorates arsenic trioxide-induced HO-1 expression and oxidative stress in THP-1 monocytes and macrophages

Lipopolysaccharide (LPS) and interferon (IFN)-gamma synergistically induced interleukin-8 (IL-8) production in human monocytic THP-1 cells. IFN-gamma-primed THP-1 cells produced higher levels of IL-8 on stimulation with LPS than non-primed cells and the level correlated with duration of priming up to 24 h, although the level of IL-8 induced was most comparable to that induced by co-stimulation with LPS and IFN-gamma. Unstimulated THP-1 cells were shown by flow cytometry to be practically devoid of membrane CD14 (mCD14). LPS and IFN-gamma enhanced mCD14 and Toll-like receptor (TLR) 4 expression in THP-1 cells, respectively, and co-stimulation with LPS and IFN-gamma induced higher levels of mCD14 and TLR4 expression than stimulation with either agent alone. LPS and IFN-gamma alone each augmented MD-2 and MyD88 mRNA expression in THP-1 cells, and co-stimulation with LPS and IFN-gamma markedly enhanced MD-2 and MyD88 mRNA expression in the cells compared to those with either LPS or IFN-gamma alone. Anti-CD14 and anti-TLR4 monoclonal antibodies almost completely inhibited IL-8 production induced by LPS plus IFN-gamma in THP-1 cells. These findings suggest that combined stimulation of THP-1 cells with LPS and IFN-gamma up-regulate mCD14, TLR4, MD-2 and MyD88 expression by these cells, which might be involved in synergistic IL-8 production by the cells.