Monocytic Cell Line THP-1 Research Articles

M2-like tumor-associated macrophages-secreted EGF promotes epithelial ovarian cancer metastasis via activating EGFR-ERK signaling and suppressing lncRNA LIMT expression

ABSTRACTBackground: Ovarian cancer (OC) is the gynecologic malignant tumor with high mortality. Accumulating evidence indicates that M2-like tumor-associated macrophages (TAMs) can secret EGF to participate in ovarian cancer growth, migration, and metastasis. An EGF-downregulated lncRNA, LIMT (lncRNA inhibiting metastasis), was identified as a critical regulator of mammary cell migration and invasion. Nevertheless, whether EGF secreted from M2-like TAMs regulates LIMT expression in ovarian cancer progression remains largely unknown.Methods: The human OC cell lines OV90 and OVCA429 were recruited in this study. The differentiation of the human monocyte cell line THP-1 into M2-like TAMs was confirmed using flow cytometry within the application of phorbol 12-myristate 13-acetate (PMA). ELISA was performed to detect EGF concentration in co-culture system of M2-like TAMs and OC cell lines. Moreover, CCK-8, flow cytometry and immunofluorescence staining of Ki67 were performed to assess the capacity of cell proliferation. Besides, cell migration and invasion were determined by wound healing and transwell assays. Furthermore, the expression levels of epithelial-mesenchymal transition (EMT) markers and EGFR/ERK signals were analyzed by qRT-PCR and western blot. Female athymic nude mice (8–12 weeks of age; n = 8 for each group) were recruited for in vivo study.Results: In the present study, THP-1 cells exhibited the phenotype markers of M2-like TAMs with low proportion of CD14+ marker and high proportion of CD68+, CD204+, CD206+ markers within the application of PMA. After co-culturing with M2-like TAMs, EGF concentration in the supernatants was significantly increased in a time-dependent manner. Besides, OC cells presented better cell viability, higher cell proliferation, and stronger migration and invasion. The expression of EMT-related markers N-cadherin, Vimentin and EGFR/ERK signals were markedly up-regulated, while E-cadherin was significantly decreased. However, these effects induced by co-culture system were reversed by the application of AG1478 (an EGFR inhibitor) or LIMT overexpression. Furthermore, the endogenous expression of LIMT was decreased in OC cell lines compared with the control group. Also, the in vivo experiments verified that the inhibition of EGFR signaling by AG1478 or overexpression of LIMT effectively repressed the tumor growth.Conclusion: Taken together, we demonstrated that EGF secreted by M2-like TAMs might suppress LIMT expression via activating EGFR-ERK signaling pathway to promote the progression of OC.

NAD metabolites interfere with proliferation and functional properties of THP-1 cells.

Over the past few years the NAD-related compounds nicotinamide (NAM), nicotinamide riboside (NR) and 1-methylnicotinamide (MNA) have been established as important molecules in signalling pathways that contribute to metabolic functions of many cells, including those of the immune system. Among immune cells, monocytes/macrophages, which are the major players of inflammatory processes, are especially susceptible to the anti-inflammatory action of NAM. Here we asked whether NAM and the two other compounds have the potential to regulate differentiation and LPS-induced biological answers of the monocytic cell line THP-1. We show that treatment of THP-1 cells with NAM, NR and MNA resulted in growth retardation accompanied by enrichment of cells in the G0/G1-phase independent of p21 and p53. NAM and NR caused an increase in intracellular NAD concentrations and SIRT1 and PARP1 mRNA expression was found to be enhanced. The compounds failed to up-regulate the expression of the cell surface differentiation markers CD38, CD11b and CD14. They modulated the reactive oxygen species production and primed the cells to respond less effectively to the LPS induced TNF-α production. Our data show that the NAD metabolites interfere with early events associated with differentiation of THP-1 cells along the monocytic path and that they affect LPS-induced biological responses of the cell line.

Evaluating the immunomodulatory potential of human amniotic epithelial cells as a therapeutic in ex vivo donor lung reconditioning

Background & Aim Ex Vivo Lung Perfusion (EVLP) provides a platform for the evaluation and reconditioning of donor lungs deemed unsuitable for transplant. Elevated expression of pro-inflammatory cytokines leading to activation of pulmonary endothelium is associated with poor outcomes after lung transplantation. EVLP offers a platform to administer advanced therapeutics, including cell-based therapies such as human Amniotic Epithelial Cells (hAECs). This study aims to evaluate the immunomodulatory properties of hAECs against macrophage and endothelial activation. Methods, Results & Conclusion hAECs were isolated from term-placenta through enzymatic digest and cultured for 4 days to generate conditioned media. Isolated cells were characterised using immunocytochemistry and flow cytometry for surface markers. The THP-1 monocytic cell line was differentiated into macrophages using phorbol 12-myristate 13-acetate (PMA) then treated with IFNγ and LPS to generate pro-inflammatory macrophages. ELISA and qPCR were used to detect any decrease in IL-8 and TNFα expression upon subsequent treatment with hAEC conditioned media. hAECs were co-cultured alongside IL-1β activated Human Pulmonary Microvascular Endothelial Cells (HPMEC), activation markers ICAM-1 and VCAM were assessed through flow cytometry. Three term-placenta were used to harvest hAECs through a serum-free process, generating large yields of viable cells. Isolated hAECs strongly expressed epithelial cell marker EpCAM (>90%) but not markers of endothelial or mesenchymal cells (≤ 2%). A significant decrease was observed for IL-8 (p=0.0001) and TNFα (p=0.004, p=0.0001) protein expression upon hAEC treatment of pro-inflammatory macrophages at 6 and 24 hours compared to the LPS control. Conditioned media treatment also leads to a 1.2-fold decrease in IL-8 gene expression compared to the LPS control at 6 hours. Mean fluorescence intensity decreased by 1.3 and 1.4-fold for ICAM-1 and VCAM respectively on hAEC treated HPMEC compared to the IL-1β control, with percentage cell expression being lowered. Correspondingly, IL-8 protein expression was significantly decreased. In this study, we demonstrated the potential for hAECs to reduce the production of key inflammatory cytokines (IL-8 and TNFα) from pro-inflammatory macrophages and activated pulmonary endothelium, alongside lowering expression of key endothelium activation markers. Future studies plan to assess the therapeutic potential of hAECs within the EVLP platform.

Preparation and characterization of CoFe2O4 and CoFe2O4@Albumen nanoparticles for biomedical applications

Biopolymer coated magnetic nanoparticles are becoming extremely popular in the fields of biomedical research because of their enormous applications potentials. In this work, first, CoFe2O4 nanoparticles (CF NPs) of two distinct sizes have been prepared following co-precipitation method by varying the synthesis parameters and characterized. XRD, SAED and IFFT (Inverse Fast Fourier Transformed) assisted HRTEM analyses confirmed the successful formation of monodispersed spinel cubic CoFe2O4 nanoparticles. Particles size of CF NPs was found to increase with increase in the amount of reducing agent. Synthesized CF NPs was coated with an egg albumen matrix by a facile and environmental friendly method to form biocompatible CoFe2O4@Albumen nanocomposite nanoparticle (CF@Alb NP). Prepared CoFe2O4 and CoFe2O4@Albumen nanoparticles were examined for its structure, morphology, thermal stability and magnetic nature employing powder XRD, HRTEM, TGA, FTIR and VSM techniques. Low as well as high magnification TEM analyses have shown coating of amorphous albumen on crystalline CF NPs. It was observed that CF@Alb NP is composed of many smaller CF NPs engulfed in the albumen matrix forming a nano-aggregate of size ∼80–130 nm. IFFT analysis of HRTEM micrograph showed presence of crystalline CF NPs in amorphous albumen matrix. Further, TGA and FTIR results also suggested the successful coating of albumen on CF NPs. CF@Alb NP has shown a very good Dox loading ability with loading efficiency of ∼93%. A promising pH dependent Dox release behavior was observed. Dox release kinetics has also been studied using different mathematical models. Biocompatibility of the CF@Alb NP has been tested against the human monocytic cell line THP-1. This novel CF@Alb NP could have a great potential in biomedical applications, particularly in hyperthermia and targeted drug delivery.

Differential induction of nuclear factor-like 2 signature genes with toll-like receptor stimulation

Inflammation is associated with production of reactive oxygen species (ROS) and results in the induction of thioredoxin (TXN) and peroxiredoxins (PRDXs) and activation of nuclear factor-like 2 (Nrf2). In this study we have used the mouse RAW 264.7 macrophage and the human THP-1 monocyte cell line to investigate the pattern of expression of three Nrf2 target genes, PRDX1, TXN reductase (TXNRD1) and heme oxygenase (HMOX1), by activation of different Toll-like receptors (TLRs). We found that, while the TLR4 agonist lipopolysaccharide (LPS) induces all three genes, the pattern of induction with agonists for TLR1/2, TLR3, TLR2/6 and TLR7/8 differs depending on the gene and the cell line. In all cases, the extent of induction was HMOX1>TXNRD1>PRDX1. Since LPS was a good inducer of all genes in both cell lines, we studied the mechanisms mediating LPS induction of the three genes using mouse RAW 264.7 cells. To assess the role of ROS we used the antioxidant N-acetylcysteine (NAC). Only LPS induction of HMOX1 was inhibited by NAC while that of TXNRD1 and PRDX1 was unaffected. These three genes were also induced by phorbol myristate acetate (PMA), a ROS-inducer acting by activation of protein kinase C (PKC). The protein kinase inhibitor staurosporine inhibited the induction of all three genes by PMA but only that of HMOX1 by LPS. This indicates that activation of these genes by inflammatory agents is regulated by different mechanisms involving either ROS or protein kinases, or both.

NF90 regulation of immune factor expression in response to malaria antigens

ABSTRACTNuclear factor 90 (NF90) is a dual DNA- and RNA-binding protein expressed ubiquitously in mammalian cells, including monocytes. Here, to elucidate the function of NF90 in the immune response, we analyzed systematically its influence on gene expression programs in the human monocytic cell line THP-1 expressing normal or reduced NF90 levels. RNA sequencing analysis revealed many mRNAs showing differential abundance in NF90-silenced cells, many of them encoding proteins implicated in the response to immune stimuli and malaria infection. The transcription of some of them (e.g. TNF, LILRB1, and CCL2 mRNAs) was modulated by silencing NF90. Ribonucleoprotein immunoprecipitation (RIP) analysis further revealed that a subset of these mRNAs associated directly with NF90. To understand how NF90 influenced globally the immune response to malaria infection, lysates of red blood cells infected with Plasmodium falciparum (iRBC lysates) or uninfected/mock-infected (uRBC lysates) were used to treat THP-1 cells as a surrogate of malaria infection. NF90 affected the stability of a few target mRNAs, but influenced more generally the translation and secretion of the encoded cytokines after treatment with either uRBC or iRBC lysates. Taken together, these results indicate that NF90 contributes to repressing the immune response in cells responding to P. falciparum infection and suggest that NF90 can be a therapeutic target in malaria.

Induction of inflammasome-dependent signalling in the human monocytic cell line THP-1 by Campylobacter lipooligosaccharides

The Campylobacter lipooligosaccharides (LOS) can stimulate membrane-bound innate immune receptors in human macrophages. However, the association of Campylobacter LOS in the stimulation of cytosolic receptors or the inflammasome remains poorly characterised. Therefore, the aim of this study was to determine the role of Campylobacter LOS in the activation of NLRP3 inflammasome-dependent signalling in a human monocytic cell line. The induction of NLRP3 inflammasome-mediated IL-1β and Caspase-1 secretion in THP-1 supernatants was quantified using ELISA following co-culture of THP-1 cells with LOS extracts from wild type C. jejuni 11168, mutant C. jejuni 11 168 with reduced LOS and two wild type C. coli strains (RM1875 and 76339). Our results demonstrate that LOS purified from both C. jejuni and C. coli can induce Caspase-1 and IL-1β production in human macrophages. However, C. jejuni 11 168 mutant LOS with modified lipid A and lack of core oligosaccharides stimulated significantly reduced Caspase-1 and IL-1β. This result was also replicated in co-culture of live wild type and mutant C. jejuni with THP-1 cells. This study provides new insight into the interaction of Campylobacter with human macrophages and suggests that variation in LOS structure may alter NLRP3 inflammasome activation.

3389 Coagulation Factor Xll-mediated contact system and its role in adaptive and innate immune cell movement

OBJECTIVES/SPECIFIC AIMS: The objectives of this study are to 1) expand upon the paradigm of HK-D5 accelerated immune cell chemotaxis; 2) characterize the role of FXII in murine models of immune-mediated disease using FXII KO mice and a small molecule inhibitor of FXIIa. METHODS/STUDY POPULATION: To test whether the addition of HK-D5 peptide would accelerate C-C chemokine receptor type 2 (CCR2)-mediated chemotaxis in vitro, a real-time transwell chemotaxis assay was developed utilizing human THP-1 monocyte cell line (Fig 1). For in vivo studies, both pharmacologic FXIIa antagonism and FXll KO mice were used. Genotyping, histopathological review, FXll protein expression, and active partial thromboplastin time (aPTT) measurements were used to phenotypically characterize FXII KO mice compared to C57BL/6 wild type mice (Fig 2). Subsequently, the FXll KO mice were studied in varied models of immune-mediated disease, including LPS-induced sepsis and experimental autoimmune encephalitis (EAE), to see if and how the absence of FXll can mitigate disease severity. The EAE model involved active immunization with myelin oligodendrocyte glycoprotein (MOG) and measurement of established clinical disease severity scores. The LPS sepsis model involved an intraperitoneal injection of LPS followed by 48-hour monitoring of core body temperature using subdermal temperature transponders as a proxy for inflammatory events related to septic shock (Fig 3). RESULTS/ANTICIPATED RESULTS: HK-D5 significantly accelerates CCR2-mediated chemotaxis compared to chemokine alone (p = 0.001) similar to HK-D5’s ability to accelerate CCR7-mediated chemotaxis as previously established. The FXll KO mice were backcrossed to the C57BL/6J background and confirmed by genotyping and complete absence of FXII protein in plasma. Compared to the control, FXII KO mice have a significantly prolonged aPTT without evidence of bleeding abnormalities, which confirms the expected phenotype previously described and recapitulates what is observed in Factor XII deficiency in humans. KO mice showed no significant gross or histopathological differences in secondary lymphoid structures compared to the control. Immunohistochemistry confirmed well-organized lymphoid structures with intact B- and T-cell populations. FXll KO mice are protected in LPS-induced septic shock and EAE models. Regarding the EAE model, FXIIa inhibition significantly reduced disease severity compared to control. In the LPS model, FXll KO mice recover within 24 hours after LPS-challenge measured subjectively and objectively by core body temperature measurement. DISCUSSION/SIGNIFICANCE OF IMPACT: The current study and previous findings suggest a novel immune signaling mechanism by which a peptide fragment of high molecular-weight kininogen (HK-D5) acts as an accelerant of both innate and adaptive immune cell chemotaxis in multiple immune contexts. This has broad implications regarding a mechanism of immune-mediated inflammation in a variety of disease states, which might be amenable to the targeting this pathway for therapeutic intent.

Proteomic changes in Mycobacterium tuberculosis H37Rv under hyperglycemic conditions favour its growth through altered expression of Tgs3(Rv3234c) and supportive proteins (Rv0547c, AcrA1 and Mpa)

Diabetes affects the presentation of tuberculosis including delayed clearance of the bacteria from host cells, however, the molecular changes which help survival of phagocytosed mycobacterium in the diabetic host are still not clear. The effect of in vitro high glucose concentrations on the proteome of the phagocytosed mycobacterium isolated from the human monocytic THP1 cell line derived macrophages has been investigated in the present study. Concurrent tuberculosis and hyperglycemia conditions were mimicked by growing M. tuberculosis infected THP1 cells under high glucose conditions. Phagocytosed bacilli were isolated 5 days post infection. Proteomics analysis of the isolated bacilli was done by two-dimensional gel electrophoresis followed by mass spectrometry. A total of 224 ± 18 protein spots were obtained out of which 10 were found to be differentially expressed under high glucose concentrations in comparison to normal glucose concentration. Further, identity of all the ten proteins namely Tgs3, Rv0547c, AcrA1, EsxU, Rv2219, Mpa, Rv2308, ORN, LucA, and Rv1414 was elucidated by peptide mass finger printing using Matrix-assisted laser desorption and ionization-mass spectrometry (MALDI/MS) assisted with MASCOT software. Though Tgs3, Rv0547c, AcrA1 and Mpa proteins have been demonstrated to play a major role in lipid metabolism under nitric oxide stress conditions, the functional role of rest of the differentially expressed proteins remains to be elucidated. Under hyperglycemic conditions in the host cells, differential expression of these proteins might help in the better survival of mycobacteria and can further act as suitable targets to design novel drugs for more effective therapy for comorbid tuberculosis and diabetes.

Alcohol dehydrogenase of Candida albicans triggers differentiation of THP-1 cells into macrophages

Candida albicans proteins located on the cell wall and in the cytoplasm have gained great attention because they are not only involved in cellular metabolism and the maintenance of integrity but also interact with host immune systems. Previous research has reported that enolase from C. albicans exhibits high immunogenicity and effectively protects mice against disseminated candidiasis. In this study, alcohol dehydrogenase (ADH) of C. albicans was cloned and purified for the first time, and this study focused on evaluating its effects on the differentiation of the human monocytic cell line THP-1. The morphological features of THP-1 cells exposed to ADH were similar to those of phorbol-12-myristate acetate-differentiated (PMA-differentiated) macrophages. Functionally, ADH enhanced the adhesion, phagocytosis, and killing capacities of THP-1 cells. A flow cytometric assay demonstrated that ADH-induced THP-1 cells significantly increased CD86 and CD11b expression. The production of IL-1β, IL-6, and TNF-α by cells increased in the presence of ADH. As expected, after pretreatment with a MEK inhibitor (U0126), ADH-induced THP-1 cells exhibited unaltered morphological features, eliminated ERK1/2 phosphorylation, prevented CD86/CD11b upregulation and inhibited pro-inflammatory cytokine increase. Collectively, these results suggest that ADH enables THP-1 cells to differentiate into macrophages via the ERK pathway, and it may play an important role in the immune response against fungal invasion.

Interferon-γ upregulates Δ42PD1 expression on human monocytes via the PI3K/AKT pathway

BackgroundWe recently identified a novel alternatively spliced isoform of human programmed cell death 1 (PD-1), named Δ42PD1, which contains a 42-base-pair in-frame deletion compared with the full-length PD-1. Δ42PD1 is likely constitutively expressed on human monocytes and down-regulated in patients infected with human immunodeficiency virus type 1 (HIV-1). The mechanism underlying the regulation of Δ42PD-1 expression in monocytes remains unknown. MethodsBy flow cytometry, we investigated the effect of Interferon-gamma (INF-γ) on the expression of Δ42PD1 in primary human monocytes as well as monocytic cell lines THP-1 and U937 cells. In addition, signaling pathway inhibitors and Δ42PD1-specific blocking antibody were used to explore the pathway involved in INF-γ-induced Δ42PD1 upregulation, and to elucidate the relationship between Δ42PD1 and TNF-α or IL-6 production by INF-γ primed monocytes in response to pre-fixed E. coli. Furthermore, we assessed T-cell proliferation, activation and cytokine production as enriched CD4+ T cells were co-cultured with THP-1 or U937 cells, with or without Δ42PD1-blocking antibody. ResultsTreatment of human peripheral blood mononuclear cells (PBMCs) with IFN-γ resulted in an approximately 4-fold increase in the expression of Δ42PD1 on monocytes. Similarly, IFN-γ upregulates Δ42PD1 expression on human monocytic cell lines THP-1 and U937, in a time- and dose-dependent manner. IFN-γ-induced Δ42PD1 upregulation was abolished by JAK inhibitors Ruxolitinib and Tasocitinib, PI3K inhibitor LY294002, and AKT inhibitor MK-2206, respectively, but not by STAT1 inhibitor and MAPK signaling pathway inhibitors. JAK, PI3K-AKT, and MAPK signaling inhibitors abolished effectively the production of TNF-α and IL-6 in INF-γ-primed monocytes in response to pre-fixed E. coli. In contrast, Δ42PD1-specific blocking antibody did not affect the IFN-γ-induced priming effect. Furthermore, the MFI ratio of Δ42PD1 to full-length PD-1 (PD-1 Δ/F ratio) was significantly and positively correlated with TNF-α (P = 0.0289, r = 0.6038) produced by circulating CD14+ monocytes in response to pre-fixed E. coli. Notably, Δ42PD1 blockage significantly inhibited CD4+ T-cells proliferation and cytokine production in the co-culture conditions. ConclusionsWe demonstrated that IFN-γ increases Δ42PD1 expression on human monocytes via activating the PI3K/AKT signaling pathway downstream of JAKs, and that the PD-1 Δ/F ratio is a potential biomarker to predict the functional state of monocytes. Notably, we revealed the Δ42PD1 play a role in T-cell regulation, providing a novel potential approach to manipulate adaptive immune response.

Requirement for Cyclic AMP/Protein Kinase A-Dependent Canonical NFκB Signaling in the Adjuvant Action of Cholera Toxin and Its Non-toxic Derivative mmCT.

Cholera toxin (CT) is widely used as an effective adjuvant in experimental immunology for inducing mucosal immune responses; yet its mechanisms of adjuvant action remain incompletely defined. Here, we demonstrate that mice lacking NFκB, compared to wild-type (WT) mice, had a 90% reduction in their systemic and mucosal immune responses to oral immunization with a model protein antigen [Ovalbumin (OVA)] given together with CT. Further, NFκB−/− mouse dendritic cells (DCs) stimulated in vitro with CT showed reduced expression of MHCII and co-stimulatory molecules, such as CD80 and CD86, as well as of IL-1β, and other pro-inflammatory cytokines compared to WT DCs. Using a human monocyte cell line THP1 with an NFκB activation reporter system, we show that CT induced NFκB signaling in human monocytes, and that inhibition of the cyclic AMP—protein kinase A (cAMP-PKA) pathway abrogated the activation and nuclear translocation of NFκB. In a human monocyte-CD4+ T cell co-culture system we further show that the strong Th17 response induced by CT treatment of monocytes was abolished by blocking the classical but not the alternative NFκB signaling pathway of monocytes. Our results indicate that activation of classical (canonical) NFκB pathway signaling in antigen-presenting cells (APCs) by CT is important for CT's adjuvant enhancement of Th17 responses. Similar findings were obtained using the almost completely detoxified mmCT mutant protein as adjuvant. Altogether, our results demonstrate that activation of the classical NFκB signal transduction pathway in APCs is important for the adjuvant action of both CT and mmCT.

Immunoregulatory properties of cell-free DNA in plasma of celiac disease patients – A pilot study

The elevated plasma cell-free DNA (cfDNA) concentrations were repeatedly reported in association with the process of inflammation. The qualitative and quantitative characteristics of plasma cfDNA in active (newly diagnosed) celiac disease patients (CD) have not yet been studied despite the fact that cfDNA of healthy individuals is able to regulate immune response. We determined the total cfDNA concentration and relative content of telomeric sequences in plasma cfDNA in CD (n = 10) and healthy age- and sex-matched controls (HC, n = 10) by quantitative PCR. To obtain the evidence that the observed biological effects are caused solely by cfDNA molecules, we applied the treatment of paired plasma samples with DNase. Using paired samples of plasma (non-treated/native and treated by DNase), we analyzed the contribution of cfDNA to the activation of TLR9 and TNF-α mRNA expression in THP1 monocytic cell line. There were no significant differences in the quantities of plasma cfDNA and relative contents of telomeric sequences in their pools. When we compared the levels of TNF-α mRNA expression in THP1 cells achieved after stimulation with native CD and HC plasma samples, we found significantly (p = .031) higher expression after stimulation with CD samples. We documented also the ability of cfDNA contained in CD plasma samples to stimulate the production of TLR9 mRNA. The TLR9 mRNA expression levels were significantly (p = .014) lowered after cfDNA removal from CD plasma samples. The design of our experiments allowed us to study the effects of cfDNA without its isolation from plasma. cfDNA contained in CD plasma samples differs significantly in its immunoregulatory capacity from cfDNA in HC plasma. The differences are caused neither by different concentrations of cfDNA in plasma samples nor by different relative abundance of telomeric sequences. Further studies are needed to elucidate the role of plasma cfDNA in celiac disease pathogenesis.

Abstract TP317: Chemokine Production by Microglia Mediates Blood-Derived Monocytes Trafficking in Neuroinflammation

Introduction: After ischemic stroke, microglia and recruited macrophages play major roles in neuroinflammation. We explored how these cells affect counterpart’s polarization and infiltration and revealed some chemokines and receptors can be important modulators of the interaction. Methods: BV2 (microglia cell line) were treated with lipopolysaccharides (LPS) or interleukin-4, and the supernatant was collected as M1 or M2 conditioned media of BV2. The supernatant of PMA differentiated THP-1 (monocyte cell line) followed by LPS treatment or interleukin-13 & IL-4co-treatment were collected as M1 or M2 conditioned media of THP-1. After BV2 or THP-1 cultured in conditioned media, the activation and polarization were assessed by ICC and confocal microscopy for CD11b, CD86 and CD206. Transwell inserts of 3 and 8 μm pore membrane were used for THP-1 infiltration and BV2 migration assay. M1/M2 conditioned media of BV2 and THP-1 and the brain tissue of ischemic mouse model were assessed by proteome profiler array (PPA) to find target cytokine and chemokine. THP-1 pretreated with antagonists of CCR2, CCR4, CXCR4 and CCR5 were used for infiltration assay, the expression of chemokine receptor in THP-1 were confirmed by western blotting. Results: Both THP-1 and BV2 expressed CD206 when cultured in M2 conditioned media of BV2 or THP-1. M2 conditioned media of BV2 increased the infiltratory ability of THP-1 while M1 conditioned media of THP-1 enhanced BV2 migration. After ischemic stroke, chemokines were significantly expressed at 3 days than at 7 days in the PPA and several chemokines such as CCL2, CCL3, CCL4 and CCL5 were match to the conditioned media of BV2 while their receptor CCR4, CCR5 shows high expression in THP-1. Antagonists of CCR2, CCR4 and CCR5 significantly reduced the infiltration ability of THP-1 to BV2 M2 conditioned media. Conclusion: CCL2, CCL3, CCL4, CCL5 and their receptors CCR4, CCR5 can be strong candidates of target proteins in new therapeutic strategies to acute brain inflammation by modulating the functions of microglia and monocytes. “This research was supported by the Brain Research Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Science, ICT & Future Planning(NRF-2016M3C7A1905098)“

Synthesis, Characterization and Cytotoxicity Evaluation on Monocytic Cell Line THP1 of Ru(II) Complexes with 1, 2-Disubstituted Benzimidazoles

Synthesis, Characterization and Cytotoxicity Evaluation on Monocytic Cell Line THP1 of Ru(II) Complexes with 1, 2-Disubstituted Benzimidazoles

ER stress differentially affects pro-inflammatory changes induced by mitochondrial dysfunction in the human monocytic leukemia cell line, THP-1.

The functional and physical interaction between mitochondria and the endoplasmic reticulum (ER) has been the subject of intense study. To test the effect of this interaction on macrophage inflammatory activation, the human macrophage-like monocytic leukemia cell line THP-1 was treated with oligomycin, rotenone, or sodium azide, which induce mitochondrial dysfunction (MD) by blocking the electron transport chain (ETC). MD induced by these agents triggered activation of various sensors and markers of ER stress. This linkage affected macrophage function since LPS-induced expression of IL-23 was enhanced by the MD inducers, and this enhancing effect was abolished by inhibition of pancreatic endoplasmic reticulum kinase (PERK) activity. This MD-mediated ER stress may be universal since it was observed in human embryonic kidney HEK293 cells and colon cancer SW480 cells. On the other hand, MD regulated LPS-induced activation of the AKT/GSK3β/β-catenin pathway in a manner not affected by inhibition of PERK or inositol-requiring enzyme 1α (IRE1α) activities. These results indicate that the occurrence of MD can lead to ER stress and these two events, separately or in combination, can affect various cellular processes.

In Vitro Infections of Macrophage-Like Cell Lines with Leishmania infantum for Drug Screening.

The study of in vitro infections is essential to evaluate distinct aspects of Leishmania biology and also invaluable for more meaningful in vitro screening of promising chemical entities. Macrophage-like cells lines from different origins are amenable to Leishmania infection. Cell lines due to their stability and standardization potential are highly valued for their capacity to support reproducible infections and consistent data. In fact, these cells have been a mainstay of leishmaniasis research for more than 40years. In this context, the human monocytic THP-1 cell line is commonly used as it can be differentiated with phorbol-12myristate-13-acetate (PMA) into macrophages that are susceptible to Leishmania infection. In this section, we will describe generalities concerning the use of cell lines for in vitro Leishmania infection using THP-1 derived macrophages and Leishmania infantum axenic amastigotes expressing luciferase associated to preclinical drug screening as example.

Characterization of sulfated polysaccharide from Laurencia obtusa and its apoptotic, gastroprotective and antioxidant activities

This study was designed to characterize the physico-chemical properties of the sulfated polysaccharide (SP) isolated from the red alga Laurencia obtusa and to evaluate its apoptotic, gastroprotective and antioxidant activities.The different macromolecular characteristics of SP were determined by size exclusion chromatography combined with multi-angle laser light-scattering detection (SEC-MALLS), Fourier transform infrared spectroscopy (FTIR) analysis and nuclear magnetic resonance spectroscopy (1H NMR and 13C NMR). The native molecular weight of the extracted polysaccharide is high (≥336,900 g·mol−1). It showed high amounts of sulfated groups (28.2%) and low levels of proteins. It was found to be a potent inducer of apoptosis on acute monocytic leukaemia THP-1cell lines with EC50 value of 53 μg·mL−1. Furthermore, a significant gastroprotective effect (p < 0.01) was also observed with a gastric ulcer inhibition of 63.44%, 78.42% and 82.15% at the doses 25, 50 and 100 mg·kg−1, respectively. In addition, SP significantly increased glutathione levels (GSH) and decreased the concentration of thiobarbituric acid-reactive substances (TBARS) in EtOH/HCl-damaged gastric mucosa in rats; it also exhibited an important antioxidant activity in vitro. Therefore, SP, derived from the red alga Laurencia obtusa, may have a potential therapeutic effect against acute myeloid leukaemia and a beneficial potential as gastroprotective and antioxidant natural product.

In vivo transcriptome changes of human white blood cells in response to vitamin D

In the vitamin D intervention study VitDbol (NCT02063334) blood samples were drawn directly before an oral bolus (2000 μg vitamin D3) and 24 h later. The focus of phase II of VitDbol was the transcriptome-wide analysis of the effects of vitamin D gene expression in human peripheral blood mononuclear cells (PBMCs). All five participants responded in an individual fashion to the bolus by increases in serum levels of the vitamin D metabolites 25-hydroxyvitamin D3 (25(OH)D3) and 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3). RNA sequencing identified 15.040 commonly expressed genes in PBMCs, 702 (4,7%) of which were significantly (p < 0,05) affected by the vitamin D3 bolus. KEGG pathway analysis suggested that these genes are involved in general protein translation, monocyte differentiation and cellular growth control. Previously published transcriptome-wide studies in comparable cell systems confirmed 234 of the 702 vitamin D target genes, leaving many genes, such as HLA-A and HLA-C, as novel discoveries. Interestingly, in vivo stimulated PBMCs of this study showed a larger number of common vitamin D target genes with the monocytic cell line THP-1 than with in vitro stimulated PBMCs. The expression pattern of vitamin D target genes differed significantly between individuals and the average expression change can serve as a marker for vitamin D responsiveness. In conclusion, this study demonstrates that under in vivo conditions changes in 25(OH)D3 and 1,25(OH)2D3 serum concentrations alter the expression of more than 700 vitamin D target genes in human leukocytes.

Forskolin inhibited the secretion of interleukin-1β in activated human THP-1 macrophages

Objective To investigate the effects of Forskolin on the activation of nucleotide-binding oligomerization domain like receptor family, pyrin domain-containing 3(NLRP-3) inflammasome and the secretion of interleukin-1β(IL-1β) in activated human THP-1 macrophages, which can provide evidence for clinical treatment of inflammatory diseases in children. Methods Human monocyte cell line THP-1 cells were induced to differentiate into macrophages by Phorbol-12-myristate-13-acetate(PMA), and Forskolin(10, 50, 100 μmol/L) stimulated activated macrophages by nigericin.The mRNA of the inflammasome markers NLRP-3, IL-1β and Caspase-1 were detected by real-time quantitative polymerase chain reaction(qPCR) method.The protein of NLRP-3, pro-IL-1β, pro-Caspase-1 and Caspase-1 were detected by Western blot.The secretion of inflammatory cytokines IL-1β was detected by enzyme-linked immuno-sorbent assay(ELISA). Results Nigericin activated the cells and the mRNA expression of NLRP-3, IL-1β and Caspase-1 increased by 7.59 times(P 0.05). Western blot showed that Forskolin also had no effect on the protein expression of pro-Caspase-1 and pro-IL-1β in activated THP-1 macrophages(P>0.05), but the expression of NLRP-3 and Caspase-1 decreased significantly (P<0.001). The results of ELISA showed that the IL-1β secretion increased from basal 584.0 nmol/L to activated 2 695.6 nmol/L(t=16.031 1, P<0.001) on THP-1 macrophages by nigericin, but Forskolin(10, 50, 100 μmol/L) reduced it to 1 858.4 nmol/L(t=5.365 5, P<0.001), 1 467.9 nmol/L(t=8.047 5, P<0.001) and 1 246.7 nmol/L(t=10.199 0, P<0.001) on the activated THP-1 macrophages. Conclusions Forskolin can not affect the expression of pro-cytokines at the gene transcription and protein translation levels in activated THP-1 cells, but inhibit the secretion of inflammatory cytokines IL-1β, so as to inhibit inflammatory response, which can be used to treat pediatric inflammatory diseases caused by IL-1β. Key words: Inflammation; Forskolin; Macrophage; Inflammasome; Interleukin-1β