Human Monocytic Leukemia Research Articles

In vitro photoprotective potential of aryl-sandwiched (thio)semicarbazones against UVA mediated cellular and DNA damage

The most prevalent solar ultraviolet radiation is ultraviolet-A (UVA) radiation. It is the inducer of reactive oxygen species (ROS), a potent mediator of inflammation and photocarcinogenesis. Regular application of sunscreens containing UVA filters is an effective preventive measure in mitigating the risk associated with the formation of dermal carcinoma. Therefore, the development of new photoprotective agents is of great need. The current work examined the in vitro photoprotection of the aryl-linked (thio)semicarbazone derivatives against UVA-mediated DNA damage, inflammation, reactive nitrogen species (RNS), and ROS. Except for the inflammatory cytokine assay, which was carried out on the human monocytic leukemia (THP-1) cell line, all tests were conducted on the human dermal fibroblast (BJ) cell line. In comparison to benzophenone (reference compound), the compound (2Z, 2′Z)-2,2′-(1,3-Phenylenebis (methanylylidene)) bis (hydrazine-1-carbothioamide) (DD-21) demonstrated considerable protection against UVA-induced damage. Compared to the UVA-irradiated control, DD-21 significantly decreased the levels of nitric oxide (NO) and ROS (p < 0.001). In the presence of DD-21, the release of UVA-induced pro-inflammatory cytokines, tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), was also significantly reduced (p < 0.05). Moreover, it was observed that DD-21 protected the cells from UVA-mediated DNA strand breaks and also inhibited the formation of cyclobutane pyrimidine dimers (CPDs) upon comparison to the UVA-exposed control cells (p < 0.001). In conclusion, the findings of this study revealed that DD-21 exhibits remarkable photoprotective properties, thus demonstrating its potential as a candidate UVA filter.

Mechanistic/mammalian target of rapamycin complex 1 (mTORC1) signaling is involved in phagocytosis activation during THP-1 cell differentiation.

Mechanistic/mammalian target of rapamycin complex 1 (mTORC1) is a serine/threonine kinase that plays a major role in cell metabolism. Although mTORC1 inhibitors are known to exert immunosuppressive effects, their effects on immune cells are not fully understood. In the present study, we examined the involvement of mTORC1 in the differentiation and functions of macrophages using THP-1 cells, which are derived from human monocytic leukemia and differentiate into macrophage-like cells upon treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA). We also examined the effects of two mTOR inhibitors, Torin 1 and rapamycin, on TPA-stimulated THP-1 cells. Although mTORC1 activation was observed upon TPA stimulation, mTOR inhibitors did not affect TPA-induced morphological changes or expression of the general macrophage marker, CD11b. In contrast, phagocytosis and fluid endocytosis were significantly impaired by the mTOR inhibitors. Endocytosis suppression was observed when mTOR inhibitors were added during differentiation, but not before or after differentiation, suggesting that endocytosis suppression altered the direction of differentiation. Furthermore, mTOR inhibitors altered the expression of M1/M2 polarization markers. These results suggest that the immunosuppressive effects of mTOR inhibitors may involve the suppression of macrophage endocytosis caused by abnormal cell differentiation.

Pyridine and isoxazole substituted 3-formylindole-based chitosan Schiff base polymer: Antimicrobial, antioxidant and in vitro cytotoxicity studies on THP-1 cells

This paper presents the synthesis of two new chitosan Schiff base (CSB) polymers, namely, 2PCT and 4MCT based on pyridin-2-ylmethyl-1H-indole-3-carbaldehyde and 1-(4-methyl-3,5-dimethylisoxazole)-1H-indole-3-carbaldehyde with chitosan (CT). The structural features of CSB polymers were confirmed by Fourier-transform infrared (FTIR) and proton nuclear magnetic resonance (1H NMR) spectroscopy and their antimicrobial activity was evaluated against Staphylococcus aureus, Escherichia coli and Candida albicans. The antioxidant studies found that both 2PCT and 4MCT presented significant free radical scavenging activity with IC50 at 169.01 and 372.84 μg/mL, respectively. The cell viability results obtained from in vitro cytotoxicity studies performed using human monocyte leukemia (THP-1) cells were found to be 75.6 ± 0.25 % and 79.1 ± 1.5 % for 2PCT and 4MCT, respectively, at a concentration of 10 mg/mL. Flow cytometry analysis demonstrated the reducing ability of CSB polymers on intracellular reactive oxygen species (ROS) in THP-1 cells. The overall results of antioxidant activity, in vitro biocompatibility and ability to reduce the intracellular ROS production emphasized that the CSB polymers prepared could serve as a potential biomaterial in biomedical applications, such as wound treatment process.

Bone mesenchymal stem cells deliver exogenous lncRNA CAHM via exosomes to regulate macrophage polarization and ameliorate intervertebral disc degeneration

Exosomes derived from bone marrow mesenchymal stem cells (BMSC-Exos) were reported to have therapeutic potential in degenerative diseases. This study aimed to explore the effects of BMSC-Exos on inhibiting M1 macrophage polarization, reducing excessive nucleus pulposus cells (NPCs) apoptosis, and inhibiting ECM degradation during intervertebral disc degeneration (IDD). Rat IDD models were established by acupuncture. For the co-culture experiment, we used BMSC-Exo or human monocyte leukemia (THP-1) medium to incubate THP-1 or NPCs, respectively. BMSC-Exo was isolated from the BMSC medium, identified by TEM and NTA, and injected into the intervertebral discs of IDD rats. The macrophage infiltration in intervertebral disc tissue was evaluated by immunohistochemistry and immunofluorescence. ELISA was used to measure the levels of TNF-α, IL-6 and IL-10. The ECM degradation was analyzed by Western blot. The cell proportion and apoptosis were measured by flow cytometry. The morphological change of the intervertebral disc was analyzed by HE and safranin O fixation staining. In intervertebral disc tissues of IDD rats, we found the increased infiltration of M1 macrophages, with upregulated iNOS, TNF-α and IL-6 levels. Compared with BMSCs, the expression of CAHM in BMSC-Exo was significantly higher. Using co-cultured experiments, we proved that BMSC-Exo reduced apoptosis and ECM degradation of NPCs by inhibiting M1-type macrophage polarization by delivering CAHM. In addition, BMSC-Exo could improve IDD in vivo, including increased proteoglycan content, reduced macrophage infiltration and ECM degradation, and decrease expression of inflammatory factors by delivering CAHM. In conclusion, BMSC-Exo delivered exogenous CAHM via exosomes to regulate macrophage polarization and ameliorate IDD.

3-Formylindole-based chitosan Schiff base polymer: Antioxidant and in vitro cytotoxicity studies on THP-1 cells

Two imine derivatives of chitosan (i-CTs), namely 2FCT and 5MCT, were synthesized by reacting chitosan (CT) with 2-(3-formyl-1H-indol-1yl)acetonitrile (2F), and 5-methoxyindole-3-carbaldehyde (5M), respectively. The antimicrobial evaluation of i-CTs exhibited stronger inhibition effect against Staphylococcus aureus, Escherichia coli and Candida albicans. The antioxidant activity of 2FCT and 5MCT showed strong scavenging ability with IC50 2.31 and 6.92 μg/mL, respectively. The results of in vitro cytotoxicity of 2FCT and 5MCT examined using human monocyte leukemia (THP-1) cells indicate no cytotoxic effect on host cells and the value of cell viability was found to be 87.08 and 84.47%, respectively. Measurement of intracellular Reactive Oxygen Species (ROS) production by flow cytometry analysis revealed that the 2FCT and 5MCT reduced the ROS generation by 83 and 43%, respectively. In summary, these findings show that i-CTs synthesized to be promising biomaterial for biomedical applications such as wound healing.

Exploiting the Anti-Inflammatory Potential of White Capsicum Extract by the Nanoformulation in Phospholipid Vesicles.

The peppers of the Capsicum species are exploited in many fields, as flavoring agents in food industry, or as decorative and therapeutic plants. Peppers show a diversified phytochemical content responsible for different biological activities. Synergic activity exerted by high levels of antioxidant compounds is responsible for their important anti-inflammatory property. A methanolic extract was obtained from a new pepper genotype and tested for anti-inflammatory activity. The extract was incorporated into phospholipid vesicles to increase the bioavailability of its bioactive components. Two types of phospholipid vesicles were produced, conventional liposomes and Penetration Enhancer containing Vesicles (PEVs). They were tested in human monoblastic leukemia U937 cell line, showing no cytotoxic effect. The intracellular reactive oxygen species (ROS) and nitric oxide (NO) levels were measured to value the in vitro efficacy of the vesicles in regulating inflammatory responses. Liposomal incorporation significantly reduced ROS levels in extract-treated LPS-activated cells. Furthermore, LC-MS/MS analyses demonstrated that liposomes facilitated the transport of the extract components across the cell membrane and their accumulation into the cytoplasm.

The effect of Kagocel® on gene expression of Toll-like receptors of innate immunity in THP-1 human monocytes with different levels of differentiation

Kagocel® is used in Russia for the treatment of viral infections. In terms of its chemical structure, Kagocel® active ingredient is a copolymer of gossypol polyphenol and carboxymethylcellulose. The study investigated antiviral and cytokine-inducing activity of Kagocel®, as well as its toxic effects. The aim of the study was to investigate the effect of Kagocel® active ingredient on the induction of expression of the innate immune system receptor genes (Toll-like receptors, TLR) in the THP-1 human acute monocytic leukemia cell line with different levels of differentiation. Materials and methods: the effect of Kagocel active ingredient was investigated at the concentrations of 0.2 and 2 mg/mL in the THP-1 human acute monocytic leukemia cell line with different levels of differentiation: non-differentiated monocytes, and monocytes differentiated into macrophage-like cells. Comparative analysis of the activity of TLR 2, 3, 4, 7, 8, 9 genes was carried out by quantitative RT-PCR. The study determined standard deviations of the levels of gene expression in the experimental cells (2deltaCq ± SD) relative to the expression in the control cells. Results: Kagocel active ingredient at the concentration of 0.2 mg/mL induced activation of TLR2 expression in THP-1 monocytes by 3.5 times, TLR3 by 2 times, TLR4 by 1.6 times, and at the concentration of 2 mg/mL also induced activation of TLR7 and TLR8 by 1.4 times, and TLR9 by 2 times. The levels of TLR2, TLR3, TLR9 induction were significantly higher in THP-1 monocytes partially differentiated into macrophage-like cells, and the highest stimulation level was observed for TLR2 (8 times). Conclusions: the results obtained characterise Kagocel® as a stimulator of TLR genes in the THP-1 cell line. The number of TLR genes induced in THP-1 monocytes was shown to increase with the increase in the product concentration. THP-1 monocyte differentiation into macrophage-like cells enhances susceptibility to Kagocel®. The positive regulation of TLR genes activity may account for antiviral and interferon-inducing properties of Kagocel®, and also suggests the possibility of expanding the use of the product for various immune-associated diseases.

Impact of cigarette versus electronic cigarette aerosol conditioned media on aortic endothelial cells in a microfluidic cardiovascular model

Atherosclerosis is a complex process involving progressive pathological events, including monocyte adhesion to the luminal endothelial surface. We have developed a functional in vitro adhesion assay using BioFlux microfluidic technology to investigate THP-1 (human acute monocytic leukaemia cell) monocyte adhesion to human aortic endothelial cells (HAECs). The effect of whole smoke conditioned media (WSCM) generated from University of Kentucky reference cigarette 3R4F, electronic cigarette vapour conditioned media (eVCM) from an electronic nicotine delivery system (ENDS) product (Vype ePen) and nicotine on monocyte adhesion to HAECs was evaluated. Endothelial monolayers were grown in microfluidic channels and exposed to 0–1500 ng/mL nicotine or nicotine equivalence of WSCM or eVCM for 24 h. Activated THP-1 cells were perfused through the channels and a perfusion, adhesion period and wash cycle performed four times with increasing adhesion period lengths (10, 20, 30 and 40 min). THP-1 cell adhesion was quantified by counting adherent cells. WSCM induced dose-dependent increases in monocyte adhesion compared to vehicle control. No such increases were observed for eVCM or nicotine. Adhesion regulation was linked to increased ICAM-1 protein expression. Staining of ICAM-1 in HAECs and CD11b (MAC-1) in THP-1 cells demonstrated adhesion molecule co-localisation in BioFlux plates. The ICAM-1 adhesion response to WSCM was downregulated by transfecting HAECs with ICAM-1 siRNA. We conclude that the BioFlux system is able to model human monocyte adhesion to primary human endothelial cells in vitro and WSCM drives the greatest increase in monocyte adhesion via a mechanism involving endothelial ICAM-1 expression.

Tantalum Particles Induced Cytotoxic and Inflammatory Effects in Human Monocytes.

The aim of this study is to evaluate the biological safety of tantalum (Ta) particles and to further explore the effects of Ta particles on human monocyte toxicity and inflammatory cytokine expression. Human monocyte leukemia (THP-1) cells were cultured with Ta and hydroxyapatite (HA) particles. Cell counting kit-8 method was used to evaluate the cytotoxicity of Ta and HA particles. The apoptosis effects were evaluated by flow cytometry, and the protein expression levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were evaluated by ELISA. The protein levels of inflammation-related signaling pathways including nuclear factor-kappa B (NF-κB) and extracellular regulated kinase (ERK) were detected by western blotting. The cytotoxicity test showed that the toxicity level of Ta in vitro was grade l, which is within the clinically acceptable range. Compared with the HA control, Ta had no significant effect on THP-1 cell apoptosis, IL-6, and TNF-α release. The phosphorylated levels of NF-κB and ERK at 3 h in the Ta group were lower than those in the HA and control groups (P < 0.001 both). These results reveal Ta particles behave good biosafety properties and provide some new insights for the future clinical use of Ta.

Effect of methoxy stilbenes\u2014analogs of resveratrol\u2014on the viability and induction of cell cycle arrest and apoptosis in human myeloid leukemia cells

The present study aimed to evaluate the cytotoxicity and its mechanism of five synthetic methoxy stilbenes, namely 3,4,4ʹ-trimethoxy, 3,4,2ʹ-trimethoxy, 3,4,2ʹ,4ʹ-tetramethoxy, 3,4,2ʹ,6ʹ-tetramethoxy, and 3,4,2ʹ,4ʹ,6ʹ-pentamethoxy-trans-stilbenes (MS), in comparison with resveratrol (RSV). Human promyelocytic (HL-60) and monocytic leukemia (THP-1) cells were treated with the tested compounds for 24 h, and cytotoxicity, cell cycle distribution, and apoptosis were evaluated. Significant differences were found in the susceptibility of these cell lines to all stilbenes, including RSV. The THP-1 cells were more resistant to cytotoxic activity of these compounds than HL-60 cells. Among the tested stilbenes, 3,4,4ʹ-tri-MS and 3,4,2ʹ,4ʹ-tetra-MS exhibited higher cytotoxicity toward both cell lines than RSV and the other methoxy stilbenes. This activity might be related to cell cycle arrest at the G2/M phase and induction of apoptosis. In this regard, 3,4,4ʹ-tri-MS and 3,4,2ʹ,4ʹ-tetra-MS at highest concentrations increased the p53 protein level particularly in HL-60 cells. Moreover, treatment with these derivatives increased the ratio of the proapoptotic Bax protein to the antiapoptotic Bcl-xl protein, suggesting the induction of apoptosis through the intrinsic mitochondrial pathway in both cell lines. Further studies are required to fully elucidate the mechanism of these activities.

Antiprotozoal investigation of three Combretum species (Combretaceae) growing in Nigeria

Summary Introduction: Combretum species has been utilised for decades in African indigenous medical practices for the treatment of several parasitic infections. Objectives: This study aims at investigating the antileishmanial, antiplasmodial and antitrypanosomal properties of Combretum racemosum, Combretum platypterum and Combretum zenkeri. Methods: The leaf extracts of the plants were screened against two strains of Plasmodium falciparum using Plasmodium lactate dehydrogenase (pLDH) assay; promastigote and amastigote forms of Leishmania donovani; and Trypanosoma brucei brucei using Alamar Blue assay. Cytotoxicity screening were also carried out on African green monkey kidney cell line (Vero) and human monocytic leukemia (THP-1) cell lines. Results: C. racemosum was active against chloroquine-sensitive (D6) and chloroquine-resistant (W2) strains of P. falciparum (IC50 of 25.6 and 26.7 µg/ml, respectively) and exerted significant antiprotozoal activities against T. brucei brucei (IC50 = 18.44 µg/ml). The extract of C. platypterum displayed a slightly lower antiplasmodial activity when compared to C. racemosum, while C. zenkeri was inactive against the parasites. In addition, the extracts failed to display significant inhibitory activity on the proliferation of L. donovani. Conclusions: This study supports the ethnomedicinal use of C. racemosum. Further research needs to be carried out to identify the antiprotozoal compounds in C. racemosum, as this could be explored for possible antiprotozoal drug development.

Depletion of the Long Non-Coding RNA MALAT1 primes Chronic Myelomonocytic Leukemia (CMML) for Differentiation Therapy with All-Trans retinoic Acid (ATRA) through the Transcription Factor CREB

Improvements in antisense technology have now enabled clinically relevant therapeutic credentialing of the noncoding genome. MALAT1 is a long non-coding RNA that, among other functions, is thought to serve as a nuclear scaffold for splicing and transcription factors. MALAT1 expression is associated with inferior prognosis across solid tumors and its depletion impairs proliferation and metastasis in preclinical solid tumor models. We found that elevated MALAT1 levels are independently associated with inferior overall survival in patients with CMML. Further, RNA-sequencing of primary CMML monocytes identified MALAT1 as the fourth most over-expressed transcript compared to controls. Therefore, we explored the biologic relevance and therapeutic candidacy of MALAT1 across several human and murine models of CMML. First, we crossed NRASQ61R/+Mx1-cre driven mice, which display a CMML-like phenotype, to MALAT1KO/KOmice. Although MALAT1KO/KOmice did not have abnormalities in complete blood counts, immunophenotyping of the hematopoietic stem cell (HSC) compartment identified statistically significantly lower numbers of HSC compared to wild type (WT) controls and a non-significant decrease in NRASQ61R/+/MALAT1KO/KOcompared to NRASQ61R/+alone. This decrease in HSC was not a result of impaired self-renewal as no differences were observed in these models after in vivo competitive transplant experiments. Therefore, we reasoned that MALAT1 expression may be controlling HSC differentiation. To test this, we transformed bone marrow cells from these models with an estrogen-regulated (ER) Hoxb8 construct enabling cells to maintain an HSC state until ER is withdrawn and myeloid differentiation is induced. ER-Hoxb8NRASQ61R/+/MALAT1KO/KOcells had increased basal levels of Gr-1 compared to ER-Hoxb8 transformed NRASQ61R/+alone that was dramatically enhanced upon ER withdrawal suggesting that MALAT1 depletion regulates myeloid differentiation. These findings were validated by assessment of morphology, transcriptome, and in vivo immunophenotyping of bone marrow and spleen cells. Further, moribund NRASQ61R/+/MALAT1KO/KOmice displayed a reduction in organomegaly typically associated with leukemic burden. We validated this in human monocytic leukemia by generating MALAT1 depleted THP-1 isogeneic cell lines using the CRISPR/Cas9 system. MALAT1 depleted THP-1 cells (MKO) had greater terminal differentiation according to immunophenotypic markers and morphology that was greatly enhanced when treated with phorbol myristate acetate. Last, MKO orthotopic xenografts demonstrated inferior human leukemia engraftment and decreased spleen and liver weights, and heterotopic xenografts exhibited reduced tumor volume, collectively suggesting diminished leukemic burden. Because ATRA has been clinically tested in CMML with minimal effects, we next explored whether MALAT1 depletion could potentiate ATRA differentiation in CMML. First, we treated MKO cells with ATRA and observed a large induction of myeloid differentiation by marker expression and morphologic assessment compared to isogenic controls. This was validated by NRASQ61R/+/MALAT1KO/KOmice demonstrating that ATRA more robustly induced differentiation compared to vehicle which was not seen in NRASQ61R/+/MALAT1+/+mice. Next, we tested MALAT1 antisense oligonucleotides (ASOs) currently under clinical development in THP-1 cells +/- ATRA and demonstrated both an increase in myeloid differentiation and apoptosis compared to ATRA alone. To test this therapeutic strategy in primary CMML specimens, we generated CMML patient-derived xenografts (n=30 mice) and treated each with ASO, ATRA, the combination, or controls and identified a more robust reduction in human HSC engraftment with the combination. To explore the mechanistic basis for these findings, we performed RNA-sequencing of MALAT1-depleted or control cells and identified that CREB target genes were differentially expressed. Basal protein levels of p-CREB were also decreased in MKO cells and were further reduced in the nucleus of MKO by western and microscopy. Lastly, overexpression of WT or constitutively active CREB but not its dominant negative rescued the differentiation effect seen in ATRA treated MKO cells. Taken together, MALAT1 is a novel, CREB-dependent regulator of myeloid differentiation and its depletion potentiates ATRA therapy. Disclosures Cluzeau: Menarini: Consultancy; Jazz Pharma: Consultancy; Abbvie: Consultancy. Komrokji:celgene: Consultancy; pfizer: Consultancy; DSI: Consultancy; JAZZ: Speakers Bureau; Novartis: Speakers Bureau; JAZZ: Consultancy; Agios: Consultancy; Incyte: Consultancy. MacLeod:Ionis Pharmaceuticals: Employment. List:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding. Epling-Burnette:Forma Therapeutics: Research Funding; Celgene Corporation: Patents &amp; Royalties, Research Funding; Incyte Corporation: Research Funding.

Establishment and characterization of a DOT1L inhibitor-sensitive human acute monocytic leukemia cell line YBT-5 with a novel KMT2A-MLLT3 fusion.

Immortalized cell lines are useful for deciphering the pathogenesis of acute leukemia and developing novel therapeutic agents against this malignancy. In this study, a new human myeloid leukemia cell line YBT-5 was established. After more than 1-year cultivation from the bone marrow of a patient with acute monocytic leukemia, YBT cell line was established. Then a subclone, YBT-5, was isolated from YBT using single cell sorting. Morphological and cytogenetical characterizations of the YBT-5 cell line were determined by cytochemical staining, flow cytometry analysis, and karyotype analysis. Molecular features were identified by transcriptomic analysis and reverse transcription-polymerase chain reaction. To establish a tumor model, 5 × 106 YBT-5 cells were injected subcutaneously in nonobese diabetic/severe combined immune-deficiency (NOD/SCID) mice. DOT1L has been proposed as a potential therapeutic target for KMT2A-related leukemia; therefore, to explore the potential application of this new cell line, its sensitivity to a specific DOT1L inhibitor, EPZ004777 was measured ex vivo. The growth of YBT-5 does not depend on granulocyte-macrophage colony-stimulating factor. Cytochemical staining showed that α-naphthyl acetate esterase staining was positive and partially inhibited by sodium fluoride, while peroxidase staining was negative. Flow cytometry analysis of YBT-5 cells showed positive myeloid and monocytic markers. Karyotype analysis of YBT-5 showed 48,XY,+8,+8. The breakpoints between KMT2A exon 10 and exon 11 (KMT2A exon 10/11) and MLLT3 exon 5 and exon 6 (MLLT3 exon 5/6) were identified, which was different from all known breakpoint locations, and a novel fusion transcript KMT2A exon 10/MLLT3 exon 6 was formed. A tumor model was established successfully in NOD/SCID mice. EPZ004777 could inhibit the proliferation and induce the differentiation of YBT-5 cells. Therefore, a new acute monocytic leukemia cell line with clear biological and molecular features was established and may be used in the research and development of new agents targeting KMT2A-associated leukemia.

Cytotoxic and immunomodulatory effects of the low concentration of titanium dioxide nanoparticles (TiO2 NPs) on human cell lines - An in vitro study

Abstract Nanoparticles (NPs) can interact with the immune system in several ways, which can either enhance or suppress the functions, depending on their characteristics and composition. Though widely used in different biomedical applications, not much is known about the immunomodulatory effects of NPs on human cell lines. In this work, the immunomodulatory effects of titanium dioxide (TiO2) nanoparticles on human monocytic leukemia (THP-1) and human mast (HMC-1) cell lines were studied in a dose-dependent manner. TiO2 NPs of different molar concentrations were synthesized by a simple sol-gel method and analyzed by X-ray diffraction (XRD), Fourier Transform Infrared (FTIR) technique, UV–vis spectroscopy, Field Emission Scanning Electron Microscopy (FE-SEM), Transmission Electron Microscopy (TEM), Dynamic Light Scattering (DLS) and Zeta Potential. The cell viability of THP-1 cells treated with TiO2 NPs was significantly reduced at higher doses as observed by MTT assay. The expressions of Pro and anti-inflammatory cytokines on THP-1 and HMC-1 cells using TiO2 NPs were studied using ELISA method. The results suggested that the secretion of cytokines (IL-6 and IL-10) on human cell lines greatly depended on the concentration of TiO2 NPs. Lower concentrations of TiO2 NPs exhibited immunomodulatory effects by inducing inflammatory responses on the treated human cell lines.

MiRNA143 Downregulated the Expression of Cyclophilin A to Inhibit the Progression of Atherosclerosis

To investigate the influence of microRNA (miR)-143 on atherosclerosis and its working mechanism via cyclophilin A. Human acute monocytic leukemia cells (human acute monocytic leukemia cells, human THP-1 monocytes) were treated with ox-LDL 80 mg/L for 48 h to establish a foam cell model. The modeling was evaluated by oil red O staining. The expressions levels of miRNA-143 and CyPA were detected by q-PCR. RNA extracted was confirmed by SiO2 nano magnetic bead. Dual-luciferase reporter gene assay was used to verify the targeted regulatory relationship between miRNA-143 and CyPA. Next, the cells were transfected with miR-143 mimics and treated with a CyPA inhibitor (cyclophilin A). The expression levels of CyPA and downstream matrix metalloproteinases-9 (MMP-9) in the downstream were detected by q-PCR and western blot, respectively. No or few lipid droplets were observed in the normal THP-1 cells. After treatment with ox-LDL for 48 h, a large amount of red-stained lipid was observed in the cytoplasm, indicating that the THP-1 derived foam cell models were successfully constructed. q-PCR results indicated that miRNA-143 was significantly downregulated in the THP-1-derived foam cell model, whereas CyPA was upregulated (P &lt; 0.05); the dual luciferase reporter gene assay was performed for miRNA-143 and 3'-UTR of CyPA, and the fluorescence intensity of the 3'-UTR vector group was significantly lower than that of the empty vector group (P &lt; 0.05). Fluorescence intensity detection of mutations at the target site showed that it had no effect was produced on the fluorescence intensity. This implied a negative regulatory effect conferred of miRNA-143 on CyPA; As compared to the model group, transfection with miRNA-143 mimics led to a significant downregulation of CyPA mRNA (P &lt; 0.05), which was accompanied by significant downregulation of both MMP-9 mRNA and protein (P &lt; 0.05). MMP-9 was also significantly downregulated upon the addition of the CyPA inhibitor cyclosporine A (P &lt; 0.05).

Antibacterial and anti‐inflammatory activities of the immune‐challenged silkworm (Bombyx mori) hemolymph with Lactobacillus cell wall extracts

AbstractInsects have been used by humans as food and traditional medicines for many years. Their antimicrobial and anti‐inflammatory activities are a main focus of medicinal insect research. In a previous study, hemocyte extracts prepared from Bombyx mori larvae were found to have anti‐inflammatory effects. Therefore, we injected Lactobacillus cell wall extracts dorsolaterally into the hemocoel to increase antimicrobial peptides produced by silkworm larva. The best overall antimicrobial activities were observed against E. coli after injection of 10 μL hemolymph from immune‐challenged larvae, although the tested extract did not affect the cell proliferation rate. The effects of the immune‐challenged B. mori hemolymph on anti‐inflammatory pathways were determined using reverse transcription quantitative polymerase chain reaction and western blotting to assess the expression of pro‐inflammatory molecules in macrophage‐like human monocytic leukemia THP‐1 cells following treatment with phorbol myristate acetate. The results demonstrated that the hemolymph of immune‐challenged silkworms had antibacterial and anti‐inflammatory effects. The effects of silkworms with immune‐challenged hemolymph could facilitate the development of new types of functional foods, feed additives, and biomaterials with antibacterial and anti‐inflammatory properties.

D-4F, an apolipoprotein A-I mimetic, suppresses IL-4 induced macrophage alternative activation and pro-fibrotic TGF-β1 expression

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.

NF‑κB inhibitor DHMEQ inhibits titanium dioxide nanoparticle‑induced interleukin‑1β production: Inhibition of the PM2.5‑induced inflammation model.

PM2.5 is a particle with a diameter <2.5µm that is often involved in air pollution. Nanoparticles <100nm are thought to invade the trachea and lungs to cause inflammation, possibly through the activation of macrophages. On the other hand, titanium dioxide (TiO2) particles can be used in models of nano‑micro‑sized particles, as one can prepare the particles with such sizes. TiO2 particles are classified into Rutile, Anatase, and Brookite types by their crystal structure. Among them, Anatase‑type TiO2 particles with a primary diameter of 50nm (A50) were reported to induce interleukin (IL)‑1β production and secretion effectively in phorbol 12‑myristate 13‑acetate‑treated human monocytic leukemia THP‑1 cells (THP‑1 macrophages). We previously designed and synthesized dehydroxymethyl‑epoxyqinomicin (DHMEQ) as an inhibitor of NF‑κB. The present study investigated whether the NF‑κB inhibitor DHMEQ inhibits TiO2 nanoparticle‑induced IL‑1β production in THP‑1 macrophages, and determined the mechanism. As a result, DHMEQ inhibited A50‑induced IL‑1β secretion in ELISA assays at nontoxic concentrations. It decreased the expression of IL‑1β mRNA, which was dependent on NF‑κB. Although NLR family pyrin domain containing 3 (NLRP3)‑inflammasome‑caspase‑1 activation is required for the maturation of IL‑1β, and DHMEQ reduced the NLRP3 mRNA expression and caspase‑1 activity; a caspase‑1 inhibitor did not influence the A50‑induced IL‑1β production. Therefore, it is likely that inhibition of pro‑IL‑1β expression by DHMEQ may be sufficient to inhibit mature IL‑1β production. Thus, DHMEQ may be useful for the amelioration of inflammation in the trachea and lungs caused by inhalation of PM2.5.

Roles of Dectin-1 in phagocytosis of Candida albicans by macrophage-like cells derived from a human acute monocytic leukemia cell line THP-1

Objective To investigate the roles of Dectin-1 in phagocytosis of Candida albicans (C. albicans) by macrophage-like cells derived from a human acute monocytic leukemia cell line THP-1. Methods THP-1 macrophage-like cells served as the target cells, and were transfected with small interfering RNA (siRNA) targeting Dectin-1 to down-regulate the expression of Dectin-1 receptor (siRNA-Dectin-1 group) . THP-1 macrophage-like cells transfected with nonsense siRNA (siRNA-NC) served as a negative control group. After transfection, the THP-1 macrophage-like cells in the above 2 groups were co-cultured with heat-killed C. albicans separately. And then, fluorescence microscopy was performed to count THP-1 macrophage-like cells phagocytosing C. albicans, and flow cytometry was used to determine the mean fluorescence intensity (MFI) of dihydrorhodamine (DHR) -123 fluorescent cells. Statistical analysis was done by one-way analysis of variance (ANOVA) and t test with the SPSS19.0 software. Results After transfection with siRNA-Dectin-1, the mRNA and protein expression of Dectin-1 significantly decreased in THP-1 macrophage-like cells (t= 26.163, P < 0.001) . After 1-, 2-, 4-hour co-culture of THP-1 macrophage-like cells with C. albicans, fluorescence microscopy showed that the phagocytosis rates of C. albicans by THP-1 macrophage-like cells were significantly lower in the siRNA-Dectin-1 group than in the negative control group (17.5% vs. 22.1%, 18.6% vs. 24.3%, 39.2% vs. 59.1%, respectively, all P < 0.05) , so were the percentage of THP-1 macrophage-like cells phagocytosing more than 3 C. albicans cells (2.2% vs. 4.7%, 2.5% vs. 5.4%, 5.1% vs. 8.3%, respectively, all P < 0.05) . After 30-minute, 1-, 2- and 4-hour co-culture of THP-1 macrophage-like cells with DHR-123-labelled C. albicans, flow cytometry showed that the MFI of C. albicans-phagocytosing cells was significantly lower in the siRNA-Dectin-1 group than in the negative control group (36.8 vs. 45.7, 54.3 vs. 62.4, 72.1 vs. 84.9, 93.6 vs. 116.7, respectively, all P < 0.05) . Conclusion Dectin-1 receptor plays an important role in the phagocytosis of C. albicans by macrophages. Key words: Candida albicans; Macrophages; Phagocytosis; Leukemia, monocytic, acute; Dectin-1

Lactobacillus fermentum UCO-979C beneficially modulates the innate immune response triggered by Helicobacter pylori infection in vitro.

Helicobacter pylori infection is associated with important gastric pathologies. An aggressive proinflammatory immune response is generated in the gastric tissue infected with H. pylori, resulting in gastritis and a series of morphological changes that increase the susceptibility to cancer development. Probiotics could present an alternative solution to prevent or decrease H. pylori infection. Among them, the use of immunomodulatory lactic acid bacteria represents a promising option to reduce the severity of chronic inflammatory-mediated tissue damage and to improve protective immunity against H. pylori. We previously isolated Lactobacillus fermentum UCO-979C from human gastric tissue and demonstrated its capacity to reduce adhesion of H. pylori to human gastric epithelial cells (AGS cells). In this work, the ability of L. fermentum UCO-979C to modulate immune response in AGS cells and PMA phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1 (human monocytic leukaemia) macrophages in response to H. pylori infection was evaluated. We demonstrated that the UCO-979C strain is able to differentially modulate the cytokine response of gastric epithelial cells and macrophages after H. pylori infection. Of note, L. fermentum UCO-979C was able to significantly reduce the production of inflammatory cytokines and chemokines in AGS and THP-1 cells as well as increase the levels of immunoregulatory cytokines, indicating a remarkable anti-inflammatory effect. These findings strongly support the probiotic potential of L. fermentum UCO-979C and provide evidence of its beneficial effects against the inflammatory damage induced by H. pylori infection. Although our findings should be proven in appropriate experiments in vivo, in both H. pylori infection animal models and human trials, the results of the present work provide a scientific rationale for the use of L. fermentum UCO-979C to prevent or reduce H. pylori-induced gastric inflammation in humans.