Purpose The aim of this research was to demonstrate the protective effects of electromagnetic field (EMF) exposure on the human microglial cell line, HMO6, against ischemic cell death induced by in vitro oxygen-glucose deprivation (OGD). Materials and methods HMO6 cells were cultured for 4 h under OGD with or without exposure to EMF with different combinations of frequencies and intensities (10, 50, or 100 Hz/1 mT and 50 Hz/0.01, 0.1, or 1 mT). Cell survival, intracellular calcium and reactive oxygen species (ROS) levels were measured. Results OGD caused significant HMO6 cell death as well as elevation of intracellular Ca²⁺ and ROS levels. Among different combinations of EMF frequencies and intensities, 50 Hz/1 mT EMF was the most potent to attenuate OGD-induced cell death and intracellular Ca²⁺ and ROS levels. A significant but less potent protective effect was also found at 10 Hz/1 mT, whereas no protective effect was found at other combinations of EMF. A xanthine oxidase inhibitor reversed OGD-induced ROS production and cell death, while NADPH oxidase and mitochondrial respiration chain complex II inhibitors did not affect cell death. Conclusions 50 Hz/1 mT EMF protects human microglial cells from OGD-induced cell death by interfering with OGD-induced elevation of intracellular Ca²⁺ and ROS levels, and xanthine oxidase is one of the main mediators involved in OGD-induced HMO6 cell death. Non-invasive treatment of EMF radiation may be clinically useful to attenuate hypoxic-ischemic brain injury.

We investigated TLR10 expression in human monocytes, THP-1 cells, cultured in hypoxia (3% O₂). Levels of both TLR10 mRNA and protein in THP-1 cells cultured in hypoxia were significantly higher than those cultured in normoxia (20% O₂). We examined intracellular reactive oxygen species (ROS) content in hypoxic cells, and TLR10 expression in cells treated with hydrogen peroxide (H₂O₂), to determine whether the increase in TLR10 expression observed with hypoxia was due to an increase in intracellular ROS levels. We found that the level of intracellular ROS in cells subject to hypoxia was significantly higher than in normoxia. Experiments with ROS synthesis inhibitors revealed that hypoxia induced ROS production is mainly due to NADPH oxidase activity. TLR10 mRNA expression was increased by treatment with H₂O₂ at concentrations ranging from 50 to 250 μM. We screened the TLR10 promoter and found putative binding sites for transcription factors (TFs), such as NF-κB, NF-AT and AP-1. Next, we examined TF activities using a luciferase reporter assay. Activities of NF-κB, NF-AT and AP-1 in the cells treated with H₂O₂ were significantly higher than in untreated cells. The experiment with TF inhibitors revealed that ROS-induced upregulation of TLR10 expression is mainly due to NF-κB activation. Overall, our results suggest that hypoxia or ROS increase TLR10 expression in human monocytes and the transcriptional activities of NF-κB are involved in this process. Therefore, it is suggested that ROS produced by various exogenous stimuli may play a crucial role in the regulation of expression and function of TLR10 as second messengers.

All-trans retinoic acid (ATRA) is an effective drug for the induction therapy of acute promyelocytic leukemia. However, the treatment is associated with adverse events such as retinoic acid syndrome (RAS) in some patients, whose histologic characteristics included organ infiltration by leukemic cells. Matrix metalloproteinase 2 (MMP-2) is often upregulated in tumor cells and plays a role in tumor cell migration and invasion by degrading the extracellular matrix. In this study, we examined the possible modulatory effects of ATRA on MMP-2 expression and secretion in human myeloid leukemia cell line THP-1. The cells were treated with various concentrations of ATRA, and MMP-2 expression and secretion were examined. MMP-2 expression and secretion started to increase with ATRA concentration as low as 0.1 nM and gradually increased thereafter. Agonists of retinoic acid receptor (RAR) or retinoid X receptor (RXR) alone could enhance MMP-2 secretion, and RAR or RXR antagonists alone could reverse ATRA-induced MMP-2 secretion. ATRA increased intracellular calcium ion levels, and a calcium-channel blocker inhibited ATRA-induced MMP-2 secretion. Dexamethasone suppressed ATRA-induced MMP-2 secretion. Our results suggest that ATRA enhances MMP-2 expression and secretion in human myeloid leukemia THP-1 cells in a calcium ion dependent manner through RAR/RXR signaling pathways, and this enhanced expression and secretion may be associated with the possible mechanisms of RAS.

All-trans retinoic acid (ATRA), an active form of vitamin A, exerts immunomodulatory functions. In this study, we examined the immune potentiating effect of ATRA on bacterial flagellin-induced NF-κB activation and proinflammatory cytokine production in human monocytic cell line THP-1. ATRA treatment significantly enhanced the flagellin-induced NF-κB/AP-1 activity in THP-1 via the RAR/RXR pathway. Similarly, ATRA enhanced the expression and production of TNF-αand IL-1βin THP-1 cells upon flagellin challenge. The cell surface expression of toll-like receptor 5 (TLR5), which is the receptor for bacterial flagellin, was significantly reduced by ATRA in a concentration- and time-dependent manner. To determine the mechanisms underlying the ATRA-enhanced immune response against bacterial flagellin despite the reduced cell surface expression of TLR5 in ATRA-treated THP-1, we examined the cell surface expression of CD14, which has been proposed to be a TLR co-receptor that enhances the response to microbial components. The cell surface expression of CD14 was significantly enhanced by ATRA treatment, especially in the presence of flagellin. Anti-CD14 antibody treatment prior to ATRA and flagellin treatments completely abolished ATRA-enhanced TNF-αand IL-1βproduction. Our results suggest that ATRA enhances flagellin-stimulated proinflammatory responses in human monocyte THP-1 cells by upregulating CD14 in a RAR/RXR-dependent manner.

Tumors highly express active heat shock protein 90 (Hsp90), which is involved in tumor survival and progression. Enhanced Toll-like receptor (TLR) 5 expression and signaling were reported to be associated with acute myeloid leukemia. In the present study, we investigated the possible modulatory effects of Hsp90 inhibitors on TLR5 expression and signaling in the human myeloid leukemia cell line THP-1. Cells were pretreated with various concentrations of the Hsp90 inhibitor geldanamycin (GA) or the Hsp70 inhibitor VER155008, followed by stimulation with bacterial flagellin. Flagellin-induced nuclear factor-κB (NF-κB) activation was significantly reduced by treatment with GA or VER155008. To elucidate the underlying mechanism of this effect, mRNA and cell surface expression of TLR5 was examined. TLR5 mRNA expression was enhanced by both GA and VER155008, whereas cell surface expression of TLR5 was reduced by three different Hsp90 inhibitors, including GA, 17-(allylamino)-17-demethoxygeldanamycin, and radicicol, and an Hsp70 inhibitor. The inhibitory effect of Hsp90 inhibitors was much higher than that of Hsp70 inhibitor. Our results suggest that Hsp90 inhibitors suppress TLR5 surface expression and activation of NF-κB in THP-1 cells in response to TLR5 ligand, and these inhibitory effects may be associated with the possible mechanisms by which Hsp90 inhibitors suppress myeloid leukemia.