Monocytic Cell Line THP-1 Research Articles

Cyclosporin A suppresses Porphyromonas gingivalis lipopolysaccharide induced matrix metalloproteinases activities in the co-culture of human gingival fibroblasts and monocyte cell line THP-1

Cyclosporine-A (CsA) is a widely used immunosuppressant. In this study, we explore the pathway through which CsA suppressed the Porphyromonas gingivalis lipopolysaccharide (P.g-LPS)-induced increase in matrix metalloproteinase (MMP) activities in co-cultured human gingival fibroblasts (HGFs) and THP-1 monocytes. In the co-culture, we found that CsA inhibited the expression of cyclophilin A (CyPA), CD147 and the activities of MMPs, which were all induced by P.g-LPS. We also found that P.g-LPS and recombinant human CyPA increased activation of ERK1/2 and IκB (an NF-κB inhibitory protein), but CsA and the anti-CD147 antibody significantly inhibited these effects. Taken together, CsA in the presence of P.g-LPS might suppress MMP activities by blocking the CyPA/CD147 interaction that results in the inhibition of ERK1/2 and NF-κB signaling by interfering with the phosphorylation of ERK1/2 and IκB.

Layer-by-Layer Self-Assembly of Polyelectrolytes on Superparamagnetic Nanoparticle Surfaces.

Designing and manufacturing multifunctional nanoparticles (NPs) are of considerable interest for both academic and industrial research. Among NPs used in this field, iron oxide NPs show low toxicity compared to metallic ones and are thus of high interest for biomedical applications. In this work, superparamagnetic Fe3−δO4-based core/shell NPs were successfully prepared and characterized by the combination of different techniques, and their physical properties were investigated. We demonstrate the efficiency of the layer-by-layer process to graft polyelectrolytes on the surface of iron oxide NPs. The influence of the polyelectrolyte chain configuration on the magnetic properties of the Fe3−δO4/polymer core/shell NPs was enlightened. The simple and fast process described in this work is efficient for the grafting of polyelectrolytes from surfaces, and thus, derived Fe3−δO4 NPs display both the physical properties of the core and of the macromolecular shell. Finally, the cytotoxicity toward the human THP-1 monocytic cell line of the core/shell NPs was assessed. The results showed that the polymer-capped Fe3−δO4 NPs exhibited almost no toxicity after 24 h of exposure at concentrations up to 25 μg mL–1. Our results show that these smart superparamagnetic nanocarriers with stealth properties are promising for applications in multimodal cancer therapy, including drug delivery.

RapaLink-1 plays an antithrombotic role in antiphospholipid syndrome by improving autophagy both in vivo and vitro

BackgroundRapaLink-1 is a third generation mammalian target of rapamycin (mTOR) inhibitor and displays superior inhibitory effect on mTOR complex 1 (mTORC1). mTOR pathway is known to block autophagy and inhibition of it can protect thrombosis-related diseases including atherosclerosis, antiphospholipid syndrome (APS) and stroke. The objective of this study was to investigate whether RapaLink-1 could exert anti-thrombotic effects on APS via improving autophagy. MethodsBALB/c mice were injected with monoclonal anti-beta-2-GPI (β2GPI) antibodies to induce APS in vivo, and anti-β2GPI antibodies together with anticardiolipin (aCL) antibodies in mice serum were assessed. The aortas of mice were isolated, and oil red and haematoxylin and eosin (HE) staining were used for thrombus morphology. The levels of LC3B and CD68 were quantified. Human monocyte cell line THP-1 was stimulated with oxidized low-density lipoprotein (ox-LDL) and treated with RapaLink-1 in vitro. The cell viability, LDH activity, apoptosis rate and rate of fate-positive cells were detected. LC3 expression was quantified by immunofluorescence. Western blot was utilized to assess the protein expression of LC3-І, LC3-П, Beclin-1 and p62. ResultsThe size of arterial thrombus plaque together with the level of anti-β2GPI antibodies and aCL was reduced by RapaLink-1. Immunostaining protocols confirmed that the application of RapaLink-1 inhibited plaque initiation and progression while decreased the extent of macrophage infiltration and enhanced the autophagy process. In vitro cultured THP-1 macrophages exposed to ox-LDL study showed that RapaLink-1 prevented cell apoptosis and enhanced autophagy of macrophages, indicated by the increasing expression of autophagy-related protein and morphological character under electron microscopy. ConclusionOur results revealed that Rapalink-1 has a potential to inhibit the formation of thrombus plaque in APS and these effects were dependent on facilitating cell autophagy both in vivo and in vitro.

Enhancer of zeste homolog 2 affects dental pulp inflammation by regulating macrophage chemotaxis

OBJECTIVE To investigate the expression changes of the epigenetic regulator enhancer of zeste homolog 2 (EZH2) during pulp inflammation and the effect of EZH2 on macrophages migration. METHODS Rat dental pulp was stimulated with 10 g/L lipopolysaccharide (LPS) to establish a model of rat pulpitis at different stages of inflammation. Immunohistochemical staining was used to detect the expression changes of EZH2 during the progression of pulp inflammation. Immunofluorescence double staining was used to detect the expression of EZH2, CD68 and their colocalization. To screen the appropriate concentration of EZH2 recombinant protein to stimulate hDPCs and human leukaemia-derived monocytic cell line (THP-1) cells, the effects of different concentrations (1, 10, 20, 40, and 100 μg/L) of EZH2 recombinant protein on proliferation of human dental pulp cells (hDPCs) and human monocyte cell line THP-1 were detected by cell counting kit-8 (CCK-8). Transwell migration assay was used to detect the effect of supernatants of hDPCs treated with EZH2 recombinant protein on the migration of THP-1 cells. RESULTS HE staining results showed that in the model of rat pulp inflammation induced by LPS, with the prolongation of LPS stimulation, the inflammation response of pulp gradually increased. Immunohistochemical results showed that EZH2 expression decreased within 8 h of LPS-induced dental pulp inflammation; but after 1, 3, and 7 d of stimulation, EZH2 expression gradually increased with the extension of the stimulation time. As for the normal rat dental pulp tissue, the positive expression of EZH2 was scattered in the odontoblast cell layer and the pulp proper. Compared with the control group, LPS stimulated the expression of EZH2 and CD68 in the infected dental pulp, and the colocalization of EZH2 and CD68 could be detected in macrophages. The results of CCK-8 suggested that the appropriate concentration of EZH2 recombinant protein to stimulate hDPCs and THP-1 cells was 20 μg/L. Transwell cell migration assay confirmed that compared with the supernatant of EZH2 untreated HDPCs group, the supernatant of EZH2treated hDPCs significantly promoted macrophage chemotaxis. CONCLUSION EZH2 is involved in the development of pulpitis and promotes the chemotaxis of macrophages, which suggests that EZH2 may play an important regulatory role in the development of pulp inflammation.

Antibacterial activity of Siparuna guianensis essential oil mediated by impairment of membrane permeability and replication of pathogenic bacteria

The increasing prevalence of resistance to conventional antibiotics in pathogenic bacteria has demanded faster development of novel sources of antibacterial agents. In this context, biological activities shown by natural compounds have received particular attention. One of the alleged backslashes for these alternative bactericidal tools is the current knowledge gap regarding their action mechanisms. Here, we evaluated the activity of essential oil extracted of Negramina, Siparuna guianensis, plants against pathogenic bacteria Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Streptococcus pyogenes. We evaluated cytotoxic effects on bacterial cells and the action mechanisms of the essential oil. The gas chromatography analysis revealed that β-Myrcene (39.68%), epicurzerenone (18.16%) and germacrenes D (14.34%) and B (2.93%) are the major components of the S. guianensis essential oil. Interestingly, the essential oil exhibited toxicity against all pathogenic bacteria without affecting the human monocytic THP-1 cell line. This antibacterial activity resulted from strong bacterial growth inhibition and deregulation of bacterial cell wall permeability with increased nucleotides and K+ ions leakage. Furthermore, our molecular docking predictions indicated high affinity between some essential oil major components Germacrene B and active sites of bacterial DNA and RNA polymerases, which indicates possible impairments on the pathogenic bacteria cell replication processes.

MiR-21-5p Under-Expression in Patients with Obstructive Sleep Apnea Modulates Intermittent Hypoxia with Re-Oxygenation-Induced-Cell Apoptosis and Cytotoxicity by Targeting Pro-Inflammatory TNF-α-TLR4 Signaling.

The purpose of this study is to explore the anti-inflammatory role of microRNAs (miR)-21 and miR-23 targeting the TLR/TNF-α pathway in response to chronic intermittent hypoxia with re-oxygenation (IHR) injury in patients with obstructive sleep apnea (OSA). Gene expression levels of the miR-21/23a, and their predicted target genes were assessed in peripheral blood mononuclear cells from 40 treatment-naive severe OSA patients, and 20 matched subjects with primary snoring (PS). Human monocytic THP-1 cell lines were induced to undergo apoptosis under IHR exposures, and transfected with miR-21-5p mimic. Both miR-21-5p and miR-23-3p gene expressions were decreased in OSA patients as compared with that in PS subjects, while TNF-α gene expression was increased. Both miR-21-5p and miR-23-3p gene expressions were negatively correlated with apnea hypopnea index and oxygen desaturation index, while TNF-α gene expression positively correlated with apnea hypopnea index. In vitro IHR treatment resulted in decreased miR-21-5p and miR-23-3p expressions. Apoptosis, cytotoxicity, and gene expressions of their predicted target genes—including TNF-α, ELF2, NFAT5, HIF-2α, IL6, IL6R, EDNRB, and TLR4—were all increased in response to IHR, while all were reversed with miR-21-5p mimic transfection under IHR condition. The findings provide biological insight into mechanisms by which IHR-suppressed miRs protect cell apoptosis via inhibit inflammation, and indicate that over-expression of the miR-21-5p may be a new therapy for OSA.

Cytotoxic and antiproliferative activities of amphibian (anuran) skin extracts on human acute monocytic leukemia cells

The use of preparations derived from frog skins for curative purposes antedates research history and is perpetuated in current medicine. The skins of anuran's (frogs and toads) are a rich source of compounds with a great importance in the search of antibiotics, analgesics, immunomodulators, enzymatic inhibitors and antitumoral agents applying to human health. Nowadays, cancer is the second most common cause of mortality with more than 8.2 million of deaths worldwide per year. Acute monocytic leukemia is the subtype M5 of acute myeloid leukemia (AML) a cancer type with reduced survival rates in patients. The monocyte to macrophage differentiation plays an essential role increasing the expansion of AML cell lines. Herein we studied the cytotoxic and antiproliferative activities of eleven amphibian species of three families belonging to Argentinean zones, against THP-1 monocytes and THP-1 macrophages acute monocytic leukemia cell lines. The evaluated species showed pronounced deleterious effects on acute monocytic leukemia THP-1 cell lines, reducing cell proliferation and inducing apoptosis, autophagy and in some cases cell aggregation. Being this work of great importance for the study of new natural anti-cancer compounds.

P002 Specific genome editing to model hypermethylation of the SP140 gene that associates to Crohn’s disease

Abstract Background SP140 (Speckled 140 KDa) encodes an epigenetic reader protein with an immune restricted expression, that binds to epigenetically modified (acetylated and methylated) histones and thereby regulates expression of large gene sets, including pro-inflammatory cytokines, in innate immune cells. SP140 is implicated in CD because single nucleotide polymorphisms, as well as defective protein function are associated with CD and marks anti-TNF response. Through a genome-wide methylation screen of Crohn’s disease (CD) patients peripheral blood, we identified two hypermethylated positions in SP140 locus associated with CD patients. We hypothesise that this DNA hypermethylation at the SP140 locus controls SP140 expression in CD patients contributing to their colitis development. Methods To address the role of SP140 DNA methylation, we used CRISPR ‘dead’ Cas9 (dCas9) epigenome-editing for specifically adding methyl groups (dCas9-DNMT) or removing methyl groups (dCas9-TET) in monocyte cell line THP1. We developed guide RNAs complementary to the gene expression regulatory region of the SP140 gene. With lentiviral delivery, we transduced THP-1 cells with guide RNA-lentiviruses, and with dCas9-DNMT or dCas9-TET lentiviruses. We assessed the level of SP140 methylation using bisulphite Sanger sequencing and the effect of methylation intervention of SP140 using qPCR and ELISA for SP140, IL-6, TNFα, IL-1β. Results We observed that SP140 gene in THP-1 cells under control conditions contained little methylated CpG sites. We induced sp140 hypermethylation through transduction of dCas9-DNMT. We validated hypermethylation of the two SP140 CpGs in transduced THP1 cells, thus mimicking the observed hypermethylation in CD patients cells. SP140 hypermethylation in THP1 cells polarised into M1 macrophages and stimulated with lipoteichoic acid (TLR-2 ligand), displayed a decrease of TNFα and (p = 0.042) protein levels. Similarly, we showed a decrease of TNFα (p = 0.02) and IL-6 (p = 0.03) protein release after transduction dCas9-DNMT and stimulation with LPS or Zymosan (TLR-2/4 ligand). Conclusion In this study, we demonstrated that editing SP140 gene methylation through CRISPR-dCAS9 technology allows modelling of the relevance of epigenetic marks for CD aetiology. Through methylome editing, we could affect the expression of CD-associated pro-inflammatory genes. Our dCas9 technique will allow us to investigate the role of DNA-methylation in the aetiology of CD.

LTA1 and dmLT enterotoxin-based proteins activate antigen-presenting cells independent of PKA and despite distinct cell entry mechanisms.

Enterotoxin-based proteins are powerful manipulators of mucosal immunity. The A1 domain of heat-labile enterotoxin from E. coli, or LTA1, is a newer adjuvant from this family under investigation for intranasal vaccines. Although LTA1 has been examined in mouse vaccination studies, its ability to directly stimulate immune cells compared to related adjuvant proteins has not been well explored. Here, we perform the first rigorous examination of LTA1’s effect on antigen presenting cells (APC) using a human monocyte cell line THP-1. To better understand LTA1’s stimulatory effects, we compared it to dmLT, or LT-R192G/L211A, a related AB5 adjuvant in clinical trials for oral or parenteral vaccines. LTA1 and dmLT both activated APCs to upregulate MHC-II (HLA-DR), CD86, cytokine secretion (e.g., IL-1β) and inflammasome activation. The effect of LTA1 on surface marker changes (e.g., MHC-II) was highly dose-dependent whereas dmLT exhibited high MHC-II expression regardless of dose. In contrast, cytokine secretion profiles were similar and dose-dependent after both LTA1 and dmLT treatment. Cellular activation by LTA1 was independent of ganglioside binding, as pre-treatment with purified GM1 blocked the effect of dmLT but not LTA1. Unexpectedly, while activation of the inflammasome and cytokine secretion by LTA1 or dmLT was blocked by the protein kinase A inhibitor H89 (similar to previous reports), these responses were not inhibited by a more specific PKA peptide inhibitor or antagonist; thus Indicating that a novel and unknown mechanism is responsible for inflammasome activation and cytokine secretion by LT proteins. Lastly, LTA1 stimulated a similar cytokine profile in primary human monocytes as it did in THP1 cells, including IL-1β, IL-6, IL-8, MIP-1α, MIP-1β, and TNFα. Thus, we report that LTA1 protein programs a dendritic cell-like phenotype in APCs similar to dmLT in a mechanism that is independent of PKA activation and GM1 binding and entry.

M2 Phenotype Macrophages Colocalize with Schwann Cells in Human Dental Pulp

Macrophages are immune cells with high plasticity that perform many functions related to tissue injury and repair. They are generally categorized as 2 functional phenotypes: M1 (proinflammatory) and M2 (anti-inflammatory and prohealing). To investigate the role of macrophages in human dental pulp, we examined the localization and distributional alterations of macrophages in healthy dental pulp as well as during the reparative process of pulp capping with mineral trioxide aggregate (MTA) and in cariously inflamed pulp of adult human teeth. We also quantified the populations of M1/M2 macrophages in healthy dental pulp by flow cytometric analysis. CD68+CD86+ cells (M1 phenotype) and CD68+CD163+ cells (M2 phenotype) were 2.11% ± 0.50% and 44.99% ± 2.22%, respectively, of 2.96% ± 0.41% CD68+ cells (pan-macrophages) in whole healthy dental pulp. Interestingly, M2 phenotype macrophages were associated with Schwann cells in healthy pulp, during mineralized bridge formation, and in pulp with carious infections in vivo. Furthermore, the M2 macrophages associated with Schwann cells expressed brain-derived neurotrophic factor (BDNF) under all in vivo conditions. Moreover, we found that plasma cells expressed BDNF. Coculture of Schwann cells isolated from human dental pulp and human monocytic cell line THP-1 showed that Schwann cells induced M2 phenotypic polarization of THP-1 cell-derived macrophages. The THP-1 macrophages that maintained contact with Schwann cells were stimulated, leading to elongation of their cell shape and expression of M2 phenotype marker CD163 in cocultures. In summary, we revealed the spatiotemporal localization of macrophages and potent induction of the M2 phenotype by Schwann cells in human dental pulp. M2 macrophages protect neural elements, whereas M1 cells promote neuronal destruction. Therefore, suppressing the neurodestructive M1 phenotype and maintaining the neuroprotective M2 phenotype of macrophages by Schwann cells may be critical for development of effective treatment strategies to maintain the viability of highly innervated dental pulp.

Effects of DNA Methylation on Leukemia Cell Proliferation.

In this study, we aimed to investigate the effect of DNA methyltransferase 1 gene (DNMT1) expression on leukemia cell proliferation. Following stimulation with interferon-α (IFN-α) or methylation inhibitor for three days, we evaluated changes in the DNMT1 expression levels, cell proliferation activity, and Bcl-2-Associated X Protein (BAX) expression levels in the chronic myelogenous leukemia cell line K562 and the acute monocytic leukemia cell line THP-1. DNMT1 expression levels and cell proliferation activity decreased in K562 and THP-1 cells, whereas BAX expression levels increased. These results suggest that the enzymatic activity of DNMT1 promotes the proliferation of tumor cells and that tumor cell proliferation can be suppressed by inhibiting DNMT1 enzymatic activity. Furthermore, because DNA methylation is associated with apoptosis, a process critical to cell growth and injury in leukemia, assessing DNMT1expression levels might help in treatment decisions for leukemia patients.

ACE overexpression in myeloid cells increases oxidative metabolism and cellular ATP

ACE overexpression in myeloid cells increases oxidative metabolism and cellular ATP

Cyclooxygenase, Lipoxygenase, Nitric Oxide Synthase, Myeloperoxidase and Protease Inhibiting Activities of the Leaves and Flowers of Mikania micrantha Kunth

Aim: To study in vitro anti-inflammatory activity of the leaves and flowers of the perennial climber Mikania micrantha. Methods: Leaves and flowers of the plant was collected in February 2015. Dried and powdered plant materials was extracted with ethyl acetate at room temperature in an orbital shaker. Cyclooxygenase, 5-lipoxygenase, myeloperoxidase and nitric oxide synthase inhibiting activities of the ethyl acetate extract were tested in human monocytic cell line THP1 after activation with lipopolysaccharide. Protease inhibiting activities were tested using trypsin as the enzyme. Percentage inhibitions of enzyme activities were calculated and IC50 values were determined. Diclofenac sodium was used as the reference drug. Results: Ethyl acetate extract of the leaves as well as flowers exhibited very good myeloperoxidase, nitric oxide synthase, cyclooxygenase and lipoxygenase inhibiting activities and showed moderate effect towards protease inhibition. The IC50 of inducible nitric oxide synthase activity was 5 and 11µg respectively for leaf and flower extracts. IC50 of myeloperoxidase activity was 4µg for flower extract. The inhibition of myeloperoxidase activity by leaf extract was so high that IC50 could not be determined at the tested concentration. IC50 of cyclooxygenase activity was 25 and 89µg and lipoxygenase activity was 98 and 185µg respectively for leaf and flower extracts. The IC50 of leaf and flower extracts towards protease inhibition were 1180 and 1250 µg respectively. Conclusion: This study revealed that Mikania micrantha contains principles which can inhibit diverse enzymes of inflammatory pathway and is a potential target for the development of new anti-inflammatory drug.

Tumor-Associated Macrophages Induce Migration of Renal Cell Carcinoma Cells via Activation of the CCL20-CCR6 Axis.

This study investigated tumor-associated macrophages activity in the microenvironment of renal cell carcinoma. Via a co-culture with macrophage-like cells differentiated from human monocyte cell line THP-1 and U937 cells, the migration ability of ACHN and Caki-1 cells, which are human renal cell carcinoma cell line cells, was significantly increased, as was the epithelial–mesenchymal transition change. A chemokine array identified the CCL20-CCR6 axis as a concentration-dependent signal in ACHN and Caki-1 cell migration. Akt in the ACHN and Caki-1 cells was activated by macrophage-like cells, and the CCL20 neutralizing antibody suppressed migration ability, epithelial–mesenchymal transition, and Akt phosphorylation in the ACHN and Caki-1 cells. Akt inhibitor AZD5363 also decreased the epithelial–mesenchymal transition change and migration ability in the ACHN and Caki-1 cells. In 42 renal cell carcinoma tissues, patients with CCR6 and macrophage infiltration indicated poor prognoses. In the tumor microenvironment of renal cell carcinoma, cancer cells are activated by CCL20 secreted by tumor-associated macrophages through Akt activation, followed by epithelial–mesenchymal transition and an acquired migration ability. Thus, inhibition of the CCL20-CCR6 axis may be a potential therapeutic strategy for renal cell carcinoma.

All-Trans Retinoic Acid Enhances Bacterial Flagellin-Stimulated Proinflammatory Responses in Human Monocyte THP-1 Cells by Upregulating CD14.

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-α and IL-1β in 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-α and IL-1β production. 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.

Soluble lytic transglycosylase SLT of Francisella novicida is involved in intracellular growth and immune suppression.

Francisella tularensis, a category-A bioterrorism agent causes tularemia. F. tularensis suppresses the immune response of host cells and intracellularly proliferates. However, the detailed mechanisms of immune suppression and intracellular growth are largely unknown. Here we developed a transposon mutant library to identify novel pathogenic factors of F. tularensis. Among 750 transposon mutants of F. tularensis subsp. novicida (F. novicida), 11 were isolated as less cytotoxic strains, and the genes responsible for cytotoxicity were identified. Among them, the function of slt, which encodes soluble lytic transglycosylase (SLT) was investigated in detail. An slt deletion mutant (Δslt) was less toxic to the human monocyte cell line THP-1 vs the wild-type strain. Although the wild-type strain proliferated in THP-1 cells, the number of intracellular Δslt mutant decreased in comparison. The Δslt mutant escaped from phagosomes during the early stages of infection, but the mutant was detected within the autophagosome, followed by degradation in lysosomes. Moreover, the Δslt mutant induced host cells to produce high levels of cytokines such as tumor necrosis factor-α, interleukin (IL)-6, and IL-1β, compared with the wild-type strain. These results suggest that the SLT of F. novicida is required for immune suppression and escape from autophagy to allow its survival in host cells.

Characterization and immunostimulatory activity of extracellular vesicles from Filifactor alocis.

Filifactor alocis, a gram-positive, obligate anaerobic rod, is an emerging periodontal pathogen that is frequently isolated from patients with periodontitis, peri-implantitis, and apical periodontitis. Recent studies have shown that extracellular vesicles (EVs) from gram-negative periodontal pathogens, so-called outer membrane vesicles (OMVs), harbor various effector molecules responsible for inducing host inflammatory responses. However, there are no reports of EVs from F. alocis. In this study, we purified and characterized the protein profiles of EVs from F. alocis and investigated their immunostimulatory activity on human monocytic THP-1 and human oral keratinocyte HOK-16B cell lines. Highly pure EVs were obtained from F. alocis using density gradient ultracentrifugation. Nanoparticle tracking analysis and transmission electron microscopy showed that F. alocis EVs were between 50 and 270nm in diameter. Proteome analysis identified 28 proteins, including lipoproteins, autolysins, F. alocis complement inhibitor (FACIN), transporter-related proteins, metabolism-related proteins, and ribosomal proteins. Human cytokine array analysis showed that F. alocis EVs remarkably induced the expression of CCL1, CCL2, MIP-1, CCL5, CXCL1, CXCL10, ICAM-1, IL-1β, IL-1ra, IL-6, IL-8, MIF, SerpinE, and TNF-α in THP-1 cells and CXCL1, G-CSF, GM-CSF, IL-6, and IL-8 in HOK-16B cells. The immunostimulatory activity of F. alocis EVs was similar to that of the whole bacterial cells. Our findings provide new insight into the role of EVs from gram-positive oral bacteria in periodontal diseases.

Interleukin-6 plays a critical role in aldosterone-induced macrophage recruitment and infiltration in the myocardium

Macrophages play an important role in aldosterone-induced myocardial fibrosis, in which the first key steps are macrophage recruitment and infiltration. We hypothesized that IL-6 may be a key mediator of aldosterone-induced macrophage recruitment and infiltration. To test this hypothesis, we designed cell studies with a human monocytic cell line THP-1 that with monocyte/macrophage functions to explore the signaling pathway of aldosterone-induced macrophage infiltration, and further investigated the phenomenon and consequent pathway in aldosterone-infused mice studies. The results showed that aldosterone induced the expression of IL-6 via mineralocorticoid receptors, and enhanced THP-1 cell migration and infiltration. Further experiments using a protease array and siRNA revealed that expressions of MMP-1 and MMP-9 were associated with aldosterone-induced macrophage infiltration. In addition, aldosterone-induced MMP-1 and MMP-9 expressions were mediated via cyclooxygenase-II and prostaglandin E2/EP-2 and EP-4 receptors. In aldosterone-infused mice, mRNA expressions of MMP-1, MMP-9 and COX-2 in peripheral blood monocytic cells were significantly increased. Moreover, the number of mouse macrophage-restricted F4/80 protein-positive cells in the myocardium was significantly higher in the aldosterone-infused mice compared with control mice. The increase in F4/80-positive cells in the myocardium was suppressed in the aldosterone-infused mice with the aldosterone antagonist eplerenone or anti-IL-6 antibody treatment. In conclusion, interleukin-6 played an important role in aldosterone-induced macrophage recruitment and infiltration in the myocardium.

LINC01140 Alleviates the Oxidized Low-Density Lipoprotein-Induced Inflammatory Response in Macrophages via Suppressing miR-23b.

Our previous study has demonstrated that miR-23b enhances oxidized low-density lipoprotein (oxLDL)-induced inflammatory response of macrophages through the A20/NF-κB signaling pathway, thus contributing to atherosclerosis. This study aims to further investigate the upstream regulators of miR-23b in mediating oxLDL-induced inflammatory response. Human monocyte cell line THP1 was induced to differentiate into macrophages followed by the oxLDL stimulation of inflammatory response. The expression of miR-23b, LINC01140, and p53 mRNA was detected by quantitative PCR. The combination of miR-23b and LINC01140 was confirmed by luciferase reporter assay and RNA immunoprecipitation. The binding of p53 and LINC01140 promoter was determined by luciferase reporter assay. The level of inflammatory cytokines, including MCP-1, TNF-α, and IL-1β, was assessed by enzyme-linked immunosorbent assay. LINC01140 was downregulated, while p53 and miR-23b were upregulated in oxLDL-induced inflammatory response of macrophages. Overexpression of LINC01140 reduced NF-κB activity by reducing miR-23b and increasing A20. The transcription of LINC01140 was inhibited by binding of p53 and the LINC01140 promoter region. Knockdown of p53 significantly reduced NF-κB activity and level of inflammatory cytokines by promoting LINC01140 expression. Our findings demonstrated that LINC01140 acts as an anti-inflammatory factor through negatively regulating miR-23/A20 axis. In addition, p53 is identified as a transcriptional repressor of LINC01140.

LncRNA NEAT1 sponges miR-214 to regulate M2 macrophage polarization by regulation of B7-H3 in multiple myeloma

BackgroundLncRNA NEAT1 was associated with the tumorigenesis of multiple myeloma (MM). However, the mechanisms of M2 macrophage polarization involved with NEAT1 in MM are still unknown. MethodsBone marrow samples, multiple myeloma cells RPMI 8226 and monocyte cell line THP-1 were used in this study. The expression of NEAT1 and miR-214 was modified by transfection with the shNEAT1 or miR-214 inhibitor. The expression of NEAT1, miR-214 and B7-H3 in MM patient tissues and cells was analyzed by RT-qPCR. ELISA assay was used to determine the release of B7-H3 in the supernatant of cell culture. The patient survival curve was analyzed using Kaplan-Meier method. The macrophage polarization markers were examined by RT-qPCR and western blotting. The interaction between NEAT1, miR-214 and B7-H3 was analyzed by Dual-Luciferase reporter and RIP assays. AG490 was used to block the JAK2/STAT3 signaling. Co-culture of THP-1 and RPMI 8226 cells was used for macrophage polarization. ResultsNEAT1 and B7-H3 were up-regulated, but miR-214 was obviously down-regulated in MM patients. B7-H3, NEAT1 and miR-214 were associated with overall survival time of MM patients. NEAT1 silencing induced miR-214 and inhibited the expression and release of B7-H3 and then suppressed M2 macrophage polarization via inhibiting the JAK2/STAT3 signaling. NEAT1 directly targeted miR-214, and miR-214 directly bound to B7-H3. MiR-214 inhibitor reversed the down-regulation and release of B7-H3 and M2 macrophage polarization caused by shNEAT1. The specific JAK2/STAT3 signaling inhibitor AG490 abrogated M2 macrophage polarization. ConclusionNEAT1 promoted M2 macrophage polarization by sponging miR-214 and then regulating B7-H3, thus accelerating MM progression via the JAK2/STAT3 signaling pathway. Our study revealed novel mechanisms of M2 macrophage polarization and provided new potential clinical therapeutic targets for MM.