Human Cell Line THP-1 Research Articles

Facilitating THP-1 macrophage studies by differentiating and investigating cell functions in polystyrene test tubes

Macrophage cell lines are a useful model to explore the properties of primary macrophages. However, a major limitation in the use of these cells is that when they are differentiated, they become adherent and hence present with the same limitation as natural macrophages. The cells need to be detached and are often subjected to detachment techniques such as detachment buffers containing proteolytic enzymes or scraping with a rubber ‘policeman’. These steps are time-consuming, reduce cell yields as well as cell viability and function. We have therefore investigated the possibility of differentiating the human macrophage THP-1 cell line in polystyrene FACS tubes to enable cells to be directly used for investigations by flow cytometry. Here we demonstrate that when the human macrophage cell line THP-1 are cultured in FACS tubes with phorbol myristate acetate added, they undergo differentiation into macrophages, assessed morphologically and by autofluorescence expression, in a similar manner to those cultured in tissue culture dishes. The cells can be readily washed and adjusted in concentration by centrifugation in the same tubes and can be directly tested for expression of cell surface markers and function by flow cytometry. This avoids the use of either detachment reagents or physical cell scraping. Consequently, we showed that the tube culture method results in increased cell yield and viability compared to those subjected to detachment procedures. The tube method generated functional macrophages which expressed the complement receptors, CR3 and CR4, and effectively phagocytosed complement opsonised Staphylococcus aureus via these receptors.

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

PO-425 The effect of bis structured schiff base material on apoptosis, cytotoxicity and DNA damage of breast cancer cell line

IntroductionHarnessing CD8 T cells to respond to tumorigenic antigens remains a supreme therapeutic strategy for sustained clearance of tumours and a resulting cancer-free life for patients. Efficient tumor-initiated T cell priming requires interferon-beta (IFN-b) production by dendritic cells and the expression of IFN-b has been demonstrated to be dependent upon activation of the Stimulator of Interferon Genes (STING) pathway. Indeed, intratumoral delivery of nucleotide-based STING agonists induces a profound regression of established tumours in syngeneic mouse models.Material and methodsHere, we describe a high-throughput screening platform for identifying non-nucleotide small molecule STING agonists. This has been established using a primary assay involving a human THP-1 cell line carrying an IRF-inducible reporter with 5 copies of the IFN signalling response element. Counter screens, involving alternative reporter constructs, rodent cell-based assays, as well as cGAS and STING knockout cell lines, are used to eliminate luciferase artefacts and ensure human-rodent cross species reactivity, as well as pathway selectivity. Biochemical assays, involving cGAS enzymatic activity and STING protein binding assays, are used to identify the specific target of identified hits.Results and discussionsTo date, from an initial screen of ~1 00 000 compounds we have identified at least one novel highly tractable STING agonist scaffold (SRCB-0001, EC50 ~1 µM), which induces expression of a type I interferon-stimulated gene signature in relevant cell types and IFN-b protein production in human peripheral blood mononuclear cells (PBMCs) with efficacy that is comparable to that observed for 2’3’-cGAMP, a natural dinucleotide STING ligand. Unlike nucleotide-based STING agonists, we have found that SRCB-0001 can be dosed orally in mice thereby lifting the restrictions of intratumoral injection as the route of administration.ConclusionThus, we propose SRCB-0074 as a first-in-class small molecule STING agonist for immuno-oncology applications.

PO-424 Discovery of a novel small molecule STING agonist as a new cancer immunotherapy

Introduction Harnessing CD8 T cells to respond to tumorigenic antigens remains a supreme therapeutic strategy for sustained clearance of tumours and a resulting cancer-free life for patients. Efficient tumor-initiated T cell priming requires interferon-beta (IFN-b) production by dendritic cells and the expression of IFN-b has been demonstrated to be dependent upon activation of the Stimulator of Interferon Genes (STING) pathway. Indeed, intratumoral delivery of nucleotide-based STING agonists induces a profound regression of established tumours in syngeneic mouse models. Material and methods Here, we describe a high-throughput screening platform for identifying non-nucleotide small molecule STING agonists. This has been established using a primary assay involving a human THP-1 cell line carrying an IRF-inducible reporter with 5 copies of the IFN signalling response element. Counter screens, involving alternative reporter constructs, rodent cell-based assays, as well as cGAS and STING knockout cell lines, are used to eliminate luciferase artefacts and ensure human-rodent cross species reactivity, as well as pathway selectivity. Biochemical assays, involving cGAS enzymatic activity and STING protein binding assays, are used to identify the specific target of identified hits. Results and discussions To date, from an initial screen of ~1 00 000 compounds we have identified at least one novel highly tractable STING agonist scaffold (SRCB-0001, EC 50 ~1 µM), which induces expression of a type I interferon-stimulated gene signature in relevant cell types and IFN-b protein production in human peripheral blood mononuclear cells (PBMCs) with efficacy that is comparable to that observed for 2’3’-cGAMP, a natural dinucleotide STING ligand. Unlike nucleotide-based STING agonists, we have found that SRCB-0001 can be dosed orally in mice thereby lifting the restrictions of intratumoral injection as the route of administration. Conclusion Thus, we propose SRCB-0074 as a first-in-class small molecule STING agonist for immuno-oncology applications.

Design and synthesis of novel 6-hydroxy-4-methoxy-3-methylbenzofuran-7-carboxamide derivatives as potent Mnks inhibitors by fragment-based drug design

A novel series of 6-hydroxy-4-methoxy-3-methylbenzofuran-7-carboxamide derivatives featured with various C-2 substituents were designed and synthesized as Mnks inhibitors through fragment-based drug design. Among them, 5b, 5i, 5o and 8k showed the best Mnk2 inhibitory activity with IC50 values of 1.45, 1.16, 3.55 and 0.27 μM, respectively. And these compounds inhibited the activity of Mnk1 at the same time. Furthermore, compounds 5o and 8k exhibited anti-proliferative effects to human leukemia cancer THP-1 and MOLM-13 cell lines and colon cancer HCT-116 cell line. Moreover, Western blot assay suggested that 8k could decrease the levels of p-eIF4E in a dose-dependent manner in HCT-116 cells. Docking studies demonstrated strong interactions between 8k and Mnk2. Therefore, this unique benzofuran scaffold demonstrated great potential to be further explored as potent Mnks inhibitors with improved potency.

Expression of interleukin-6 in cystic lesions of patients with acne vulgaris and in vitro effect of Propionibacterium acnes on the production of interleukin-6 by human THP-1 monocytes

Objective To determine the expression of interleukin-6 (IL-6) in cystic lesions of patients with acne vulgaris, and to evaluate the in vitro effect of Propionibacterium acnes (P. acnes) on the production of IL-6 and activation of p38 mitogen-activated protein kinase (p38MAPK) in the human acute monocytic leukemia cell line THP-1. Methods Real-time fluorescence-based quantitative PCR was performed to determine the mRNA expression of IL-6 in cystic lesions of 6 patients with acne vulgaris, as well as in skin tissues of 6 healthy persons. Some cultured THP-1 cells were divided into 5 groups to betreated with 2 × 106 CFU/ml, 2 × 107 CFU/ml and 2 × 108 CFU/ml heat-killed P. acnes suspensions (P. acnes groups) , 100 μg/L lipopolysaccharide (LPS group) and RPMI 1640 medium (control group) respectively. After 1-, 3- and 6-hour treatment, real-time fluorescence-based quantitative PCR was conducted to determine the mRNA expression of IL-6 in the above groups. Enzyme-linked immunosorbent assay (ELISA) was performed to detect the level of IL-6 in the culture supernatant of cells in the 2 × 108-CFU/ml P. acnes group, LPS group and control group at 24 hours after the treatment. Western blot analysis was conducted to determine the protein expression of p38MAPK and phosphorylated p38MAPK in the 2 × 108-CFU/ml P. acnes group after 15-, 30- and 60-minute treatment, as well as in the LPS group after 30-minute treatment and in the control group. Some other THP-1 cells were divided into 3 groups: 2 × 108-CFU/ml P. acnes group treated with 2 × 108 CFU/ml P. acnes suspensions, SB203580 (an inhibitor of p38MAPK) group treated with 20 μmol/L SB203580 for 30 minutes followed by the treatment with 2 × 108 CFU/ml P. acnes suspensions, and control group treated with RPMI 1640 medium alone. After 6-hour treatment, the mRNA expression of IL-6 in the above 3 groups was measured by real-time fluorescence-based quantitative PCR. Results The mRNA expression of IL-6 was significantly higher in the cystic lesions of acne vulgaris than in the normal skin tissues (3.680 ± 0.790 vs. 1.155 ± 0.250, t= 3.047, P < 0.05) . Two-way analysis of variance showed that there were significant difference in the mRNA expression of IL-6 among the 2 × 106-CFU/ml, 2 × 107-CFU/ml and 2 × 108-CFU/ml P. acnes groups, LPS group and control group (F= 532.3, P < 0.001, v= 4) , and the mRNA expression of IL-6 significantly differed among different time points (F= 526.6, P < 0.001, v= 2) . There were also significant differences in the IL-6 level in the culture supernatant of cells among the 2 × 108-CFU/ml P. acnes group ([1 618.22 ± 32.23]ng/L) , LPS group ([3 212.06 ± 353.00]ng/L) and control group ([147.10 ± 0.53]ng/L; v= 2, F= 102.35, P < 0.01) . After 15-, 30- and 60-minute treatment with 2 × 108 CFU/ml P. acnes suspensions, the protein expression of phosphorylated p38MAPK obviously increased. The mRNA expression of IL-6 in THP-1 cells was significantly lower in the SB203580 group than in the 2 × 108-CFU/ml P. acnes group (t= 15.91, P= 0.004) . Conclusions The mRNA expression of IL-6 evidently increases in the cystic lesions of patients with acne vulgaris. P. acnes can activate the signaling molecule p38MAPK in THP-1 cells, and promote the production of IL-6 by THP-1 cells. Key words: Acne vulgaris; Propionibacterium acnes; Interleukin-6; Leukemia, monocytic, acute; p38 Mitogen-activated protein kinases

The microRNA-1908 up-regulation in the peripheral blood cells impairs amyloid clearance by targeting ApoE.

To give a new insight into the mechanism of ApoE dysregulation and microRNA-1908 in Alzheimer's disease (AD). Plasma ApoE levels were measured in 20 AD patients and 20 healthy controls. THP-1 was maintained in RPMI1640 with 10% fetal bovine serum. Quantitative real-time polymerase chain reaction was performed to detect 13-microRNA and ApoE mRNA in cultured cell lines. Enzyme-linked immunosorbent assay was used to measure human ApoE in the plasma or culture medium of cell lines and also used to quantify the human Aβ42 in the culture medium of cell lines. We found plasma ApoE level reduced in AD patients (2.28 vs 3.78μg/mL, P<.001), and microRNA-1908 was up-regulated in AD patients and was negatively associated with plasma ApoE (r=-0.32, P=.012). In human macrophage cell line THP-1 and astrocytoma cell line U87, microRNA-1908 could inhibit the mRNA and protein levels of ApoE by targeting its 3'untranslated region. Consistently, microRNA-1908 inhibits the ApoE-mediated Aβ clearance. Our study provides new insight into the mechanism of ApoE dysregulation in AD patients, and microRNA-1908 might be a therapeutic target for AD treatment.

Anti-inflammatory effects of α-linolenic acid in M1-like macrophages are associated with enhanced production of oxylipins from α-linolenic and linoleic acid

Chronic inflammation, mediated in large part by proinflammatory macrophage populations, contributes directly to the induction and perpetuation of metabolic diseases, including obesity, insulin resistance and type 2 diabetes. Polyunsaturated fatty acids (PUFAs) can have profound effects on inflammation through the formation of bioactive oxygenated metabolites called oxylipins. The objective of this study was to determine if exposure to the dietary omega-3 PUFA α-linolenic acid (ALA) can dampen the inflammatory properties of classically activated (M1-like) macrophages derived from the human THP-1 cell line and to examine the accompanying alterations in oxylipin secretion. We find that ALA treatment leads to a reduction in lipopolysaccharide (LPS)-induced interleukin (IL)-1β, IL-6 and tumor necrosis factor-α production. Although ALA is known to be converted to longer-chain PUFAs eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), DHA oxylipins were reduced overall by ALA treatment, as was LPS-induced secretion of EPA oxylipins. In contrast, we observed profound increases in oxylipins directly derived from ALA. Lipoxygenase products of linoleic acid were also dramatically increased, and LPS-induced production of AA oxylipins, particularly prostaglandin D2, was reduced. These results suggest that ALA may act to dampen the inflammatory phenotype of M1-like macrophages by a unique set of mechanisms distinct from those used by the long-chain omega-3 fatty acids EPA and DHA. Thus, there is strong rationale for investigating the functions of ALA oxylipins and lesser-known LA oxylipins since they hold promise as anti-inflammatory agents.

RNase 7 but not psoriasin nor sPLA2-IIA associates with Mycobacterium tuberculosis during airway epithelial cell infection.

Tuberculosis is a disease caused by Mycobacterium tuberculosis (Mtb). Innate immunity is the first line of defense against Mtb and malfunctions in any of its components are associated with the susceptibility to the disease. Epithelial products such as host defense peptides (HDPs) are the first molecules produced to counteract the infection. Although a wide variety of HDPs are produced by epithelial cells only a few of them have been studied during Mtb infection. Here, we assessed the expression and production of the HDPs psoriasin, secreted phospholipases A2 (sPLA2-IIA) and Ribonuclease (RNase) 7 in airway epithelial cells (NCI-H292), type II pneumocytes (A549 cells) and monocyte-derived macrophages from human peripheral blood mononuclear cells and from the human cell line THP1 after Mtb in vitro infection. Results show that psoriasin and sPLA2-IIA were not induced by Mtb in any of the evaluated cells, while RNase 7 was overexpressed in infected airway epithelial cells. Intracellular analysis by flow cytometry demonstrated that the highest levels of RNase 7 were observed 6 h post-infection and the induction was dependent on direct interaction between airway epithelial cells and Mtb. In addition, analysis by electron microscopy showed that RNase 7 was capable of attaching to the cell wall of intracellular mycobacteria. Our studies suggest that the induction of RNase 7 in response to Mtb could have a role in anti-mycobacterial immunity, which needs to be studied as an innate immune mechanism.

Overview of the Biological Activities of a Methanol Extract from Wild Red Belt Conk, Fomitopsis pinicola (Agaricomycetes), Fruiting Bodies from Central Italy.

Fomitopsis pinicola (Sw.) P. Karst. (Fomitopsidaceae) is a medicinal mushroom with a variety of healthy properties. In this study we tested the radical scavenging activity and antimicrobial and anticancer potential of methanol extracts of F. pinicola from central Italy. Molecular identification confirmed that the samples were F. pinicola; a Basic Local Alignment Search Tool search showed a close match (99% sequence identity) with European isolates of this species. The free radical scavenging capacities, measured by DPPH assay, showed that the extract activity was 3.5% that of Trolox. The MTT test, evaluated after 72 hours of treatment with increasing doses of extract (5-500 μg · mL-1), considerably inhibited proliferation in a dose-dependent manner in 2 human tumor cell lines. This reduction was coupled with a relevant induction of apoptosis in the human leukemia THP-1 cell line after 24 hours of treatment, but a relevant toxic effect occurred in the human colon adenocarcinoma HT29 cell line. The genotoxic potential of the methanol extracts was studied by single-cell gel electrophoresis of normal human leukocytes exposed to 20 μg extract at 37°C for 30 minutes; no DNA damage was observed. The F. pinicola methanol extract was found to have varying degrees of antifungal effects against the pathogenic fungi tested (minimum inhibitory concentration from 23.63 to 66.81 μg · mL-1). The results show that the tested F. pinicola extract has strong antimicrobial and chemo-preventive activities, but is a poor antioxidant.

Andrographolide inhibits chikungunya virus infection by up-regulating host innate immune pathways

Objective: To investigate the therapeutic efficacy of andrographolide, a plant derived compound, against chikungunya virus (CHIKV) infection. Methods: Using flow cytometry and immunoblotting assay, in vitro viral protein expression was studied in THP-1 cells line. In Balb/c mouse neonates, viral RNA copy number was determined by real time PCR. Results: The results showed reduced CHIKV protein expression on andrographolide treatment in CHIKV-infected human peripheral blood mononuclear cells, Vero cells and THP-1 cell line. In vivo, andrographolide treatment to CHIKV-infected neonates reduced viral RNA copy number. Further, andrographolide also increased cytotoxic T lymphocytes both in vitro and in vivo. Andrographolide also activated host innate immune pathways, viz., protein kinase R, phosphorylated eukaryotic initiation factor 2α , retinoic acid inducible gene-I and interferon regulatory factor 3/7, thereby increasing IFN- α secretion. CHIKV-induced nuclear factor κ light chain enhancer of activated B cells and tumor necrosis factor- α was also reduced on andrographolide treatment. Conclusion: Andrographolide inhibits CHIKV by suppressing viral protein expression and up-regulating host innate immunity and hence could be an effective therapeutic agent against CHIKV infection.

Polyacetylenes from the florets of Carthamus tinctorius and their cytotoxicity

Two new polyacetylenes, (2R,11S,12S)-(3E)-tridecene-5,7,9-triyne-1,2,11,12-tetraol (1) and (8S)-deca-4,6-diyne-1,8-diol-8-O-β-d-glucopyranoside (2), together with six known polyacetylenes (3–8), were isolated from the florets of Carthamus tinctorius. Their structures were elucidated on the basis of spectroscopic data (NMR and HRMS) and Mo2(OAc)4-induced circular dichroism. Compounds 4 and 5 were cytotoxic to the human cancer cell lines HL-60 (IC50 = 6.40, 3.66 μM), THP-1 (IC50 = 8.67, 6.69 μM) and PC-3 (IC50 = 11.30, 10.92 μM), comparable to the positive control, fluorouracil (IC50 = 2.58, 3.95 and 22.35 μM, respectively).

ATIVIDADE CITOTÓXICA DO EXTRATO DE CROTON URUCURANA BAILL CONTRA LINHAGENS DE CÉLULAS LEUCÊMICAS HUMANAS U937 E THP1

Leukemias are malignant neoplasms that affect blood cells and originate in bone marrow. These are cancers of high incidence and are difficult to treat and there is a constant search for more efficient therapies. The present work investigated the cytotoxic action of the hydroalcoholic extract of Croton urucurana in human leukemia cells U937 and THP-1. The U937 and THP-1 cell lines were plated in a 100 μL/well volume (1x106 cells/mL) in 96-well plates, treated with extracts at final concentrations of 50 μg/mL, 100 μg/mL, 200 μg/mL and 400 μg/mL and 800 μg/mL for cell viability tests with MTT(-3- (4.5-dimethyl-2-thiazole) 2.5-diphenyl-2H-tetrazolium bromide). After 48h, viability was assessed by MTT and the supernatant was used for determination of the Lactate Dehydrogenase (LD) enzyme. Fluorescence microscopy was used to evaluate apoptosis. The extract was able to reduce cell viability by the MTT assay, increase LD release and induce apoptosis in both cell lines tested at concentrations of 400 and 800 μg/ mL (p≤0.05). The C. urucurana extract presented cytotoxic activity against human leukemic cell lines U937 and THP-1, presenting itself as promising in the search for new antineoplastic agents.

Chlorflavonin Targets Acetohydroxyacid Synthase Catalytic Subunit IlvB1 for Synergistic Killing of Mycobacterium tuberculosis.

The flavonoid natural compound chlorflavonin was isolated from the endophytic fungus Mucor irregularis, which was obtained from the Cameroonian medicinal plant Moringa stenopetala. Chlorflavonin exhibited strong growth inhibitory activity in vitro against Mycobacterium tuberculosis (MIC90 1.56 μM) while exhibiting no cytotoxicity toward the human cell lines MRC-5 and THP-1 up to concentrations of 100 μM. Mapping of resistance-mediating mutations employing whole-genome sequencing, chemical supplementation assays, and molecular docking studies as well as enzymatic characterization revealed that chlorflavonin specifically inhibits the acetohydroxyacid synthase catalytic subunit IlvB1, causing combined auxotrophies to branched-chain amino acids and to pantothenic acid. While exhibiting a bacteriostatic effect in monotreatment, chlorflavonin displayed synergistic effects with the first-line antibiotic isoniazid and particularly with delamanid, leading to a complete sterilization in liquid culture in combination treatment. Using a fluorescent reporter strain, intracellular activity of chlorflavonin against Mycobacterium tuberculosis inside infected macrophages was demonstrated and was superior to streptomycin treatment.

New ferrocenyl guanidines as potent antioxidants, protein kinase inhibitors and cytotoxic agents against human leukemia THP-1 cell line

Six new ferrocenyl guanidines were synthesized and characterized by elemental analysis, FT-IR and 1H and 13C NMR. Compounds 1–6 were screened for antioxidant, protein kinase inhibition, lethality of brine shrimp, and cytotoxicity against the human leukemia THP-1 cell line. The compounds demonstrated moderate to significant activities. Antioxidant activity of the synthesized compounds was evaluated by DPPH % inhibition with IC50 values. All compounds 1–6 showed notable antioxidant activity with DPPH having IC50 values between 23.2–15.1 and noteworthy brine shrimp lethality. Compound 6 demonstrated the highest cytotoxicity against brine shrimps with LC50 of 9.09 μg/mL. Protein kinase inhibition activity showed significant hyphea formation inhibition at 100 μg/disc with 10 to 13 mm clear zone of inhibition for the compounds 2–6. The compounds were screened for in vitro cytotoxicity using human leukemia cell line (THP-1 ATCC#TIB-202). Among all compounds, the most significant activity was demonstrated by compounds 6 and 5 with IC50 of 3.88 and 5.59 μg/mL which was comparable with the standard 5-flourouracil and vincristine drugs.

In Vitro and In Vivo Methods for Analysis of Nanoparticle Potential to Induce Delayed-Type Hypersensitivity Reactions.

Delayed-type hypersensitivity (DTH) reactions are among the common reasons for drug withdrawal from clinical use during the post-marketing stage. Several in vivo methods have been developed to test DTH responses in animal models. They include the local lymph node assay (LLNA) and local lymph node proliferation assay (LLNP). While LLNA is instrumental in testing topically administered formulations (e.g., creams), the LLNP was proven to be predictive of drug-mediated DTH in response to small molecule pharmaceuticals. Global efforts in reducing the use of research animals lead to the development of in vitro models to predict test-material-mediated DTH. Two such models include analysis of surface marker expression in human cell lines THP-1 and U-937. These tests are known as the human cell line activation test (hCLAT) and myeloid U937 skin sensitization test (MUSST or U-SENS), respectively. Here we describe experimental procedures for all these methods, discuss their in vitro-in vivo correlation, and suggest a strategy for applying these tests to analyze engineered nanomaterials and nanotechnology-formulated drug products.

A neuropeptide, Substance-P, directly induces tissue-repairing M2 like macrophages by activating the PI3K/Akt/mTOR pathway even in the presence of IFN\u03b3

Macrophage polarization plays an important role in tissue damage and repair. In this study, we show that Substance-P (SP) can directly induce M2 polarization of inflammatory macrophages. SP induced the differentiation of GM-CSF-differentiated pro-inflammatory macrophages into alternatively activated phagocytic M2 like macrophages (M2SP) through direct activation of the PI3K/Akt/mTOR/S6kinase pathway and induction of Arginase-1, CD163, and CD206, all of which were nullified by pretreatment with the neurokinin-1 receptor (NK-1R) antagonist RP67580 and specific signaling pathway inhibitors. M2SP were distinct from IL-4/IL-13-induced M2a and IL-10-induced M2c subtypes; they did not show STAT activation and exhibited high phagocytic and endothelial adhesive activity. Furthermore, SP had a dominant effect on M2 polarization over Interferon gamma (IFNγ), a potent M1-skewing cytokine, and effectively induced the M2 phenotype in monocytes and the human THP-1 cell line. Finally, adoptively transferred M2SP migrated to a spinal cord injury (SCI) lesion site and improved functional recovery. Collectively, our findings show that SP, a neuropeptide, plays a role as a novel cytokine by inducing tissue-repairing M2SP macrophages and thus may be developed for pharmacological intervention in diseases involving chronic inflammation and acute injury.

Effects of the yeast form of Sporothrix schenckii on activation of p38MAPK and expression of interleukin-6 in human THP-1 macrophage-like cells

Objective To evaluate effects of the yeast form of Sporothrix schenckii on activation of p38 mitogen-activated protein kinase (p38MAPK) and expression of interleukin-6 (IL-6) in macrophage-like THP-1 cells, which were differentiated from the human acute monocytic leukemia cell line THP-1. Methods THP-1 macrophage-like cells were divided into 3 groups to be treated with the yeast form of Sporothrix schenckii at a concentration of 2 × 106 colony-forming units (CFU) /ml (yeast form group) , 100 mg/L curdlan (curdlan group) and RPMI 1640 medium (blank control group) respectively. Real-time fluorescence-based quantitative PCR was performed to measure the mRNA expression of IL-6 in THP-1 macrophage-like cells in the above 3 groups after 3-and 6-hour treatment separately, and enzyme-linked immunosorbent assay (ELISA) to detect the level of IL-6 in the culture supernatant of THP-1 macrophage-like cells after 24-hour treatment. Western blot analysis was conducted to determine the protein expression of p38MAPK and phosphorylated p38MAPK (p-p38MAPK) in the above 3 groups after 30-and 60-minute treatment separately. Other THP-1 macrophage-like cells were pretreated with 100 nmol/L dexamethasone (a p38MAPK inhibitor) for 30 minutes, and then were divided into 3 groups to be treated with the yeast form of Sporothrix schenckii, curdlan and RPMI 1640 medium respectively, and changes in the level of p-p38MAPK and mRNA expression of IL-6 were also detected. Statistical analysis was carried out with SPSS19.0 software by using one-way or multi-way analysis of variance and least significant difference (LSD) test. Results Significant differences in the mRNA expression of IL-6 in THP-1 macrophage-like cells were observed among the yeast form group, curdlan group and blank control group (F= 5 552.22, P < 0.001) after 3-hour treatment (56.81 ± 7.36, 26.69 ± 1.22 and 0.97 ± 0.05, respectively) and 6-hour treatment (378.03 ± 16.67, 276.24 ± 39.13 and 1.02 ± 0.04, respectively) . Additionally, the yeast form group showed significantly higher mRNA expression of IL-6 after 6-hour treatment than that after 3-hour treatment (q= 16.74, P < 0.001) . After 24-hour treatment, the level of IL-6 in the culture supernatant of THP-1 macrophage-like cells also significantly differed among the yeast form group, curdlan group and blank control group (59.96 ± 18.16 pg/L, 91.01 ± 17.27 pg/L, 5.50 ± 2.30 pg/L, respectively; F= 26.62, P < 0.01) , and was significantly higher in the yeast form group than in the blank control group (P < 0.01) . After 30-and 60-minute treatment, the protein expression of p-p38MAPK was significantly higher in the yeast form group than in the blank control group (both P < 0.01) . Moreover, the mRNA expression of IL-6 (4.46 ± 1.03 vs. 493.52 ± 113.87, P < 0.001) and protein expression of p-p38MAPK (2.29 ± 0.37 vs. 4.55 ± 0.46, q= 10.81, P < 0.01) were both significantly lower in the yeast form group with dexamethasone pretreatment than in that without dexamethasone pretreatment. Conclusion In vitro treatment with the yeast form of Sporothrix schenckii can enhance the expression of IL-6 in human THP-1 macrophage-like cells by activating the p38MAPK signaling pathway. Key words: Sporothrix; Macrophages; Interleukin-6; p38 Mitogen-activated protein kinases

Lipid Nanostructures Containing Atorvastatin Decrease Lipopolysaccharide-Induced Inflammation in Macrophages

Atorvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, is known to exert lipid-lowering, but also anti-inflammatory effects in extra-arterial locations. In order to avoid its toxicity associated with long-term oral treatment, in this study we have proposed novel lipid nanostructures containing atorvastatin to improve its efficiency and bioavailability after local application in periodontitis inflammation therapy. The physico-chemical characterization of the nanostructures was performed using dynamic light scattering technique and morphological observations were made by light microscopy. The encapsulation efficiency was determined by high performance liquid chromatography analysis of loaded atorvastatin. In vitro cytotoxicity and anti-inflammatory activity were evaluated in human premonocytic THP-1 cell line and a model of lipopolysaccharide-induced inflammation in macrophages, respectively. The results showed that the population of atorvastatin lipid nanostructures presented a mean diameter of 178 nm and a good homogeneity after sonication and extrusion treatments applying, as indicated by the low polydispersity index of 0.223. The efficiency of atorvastatin encapsulation was high (87.3%) and the nanostructures cytotoxicity was reduced for lipid concentrations ranging from 50 μM to 500 μM. Experiments in THP-1 cells differentiated to macrophages demonstrated that atorvastatin liposomal formulation led to a higher inhibition of lipopolysaccharide-induced proinflammatory cytokines (interleukin 6, tumor necrosis factor alpha and interleukin 8) release, compared to free drug. In conclusion, atorvastatin lipid nanostructures could be used to develop an efficient local treatment of periodontitis inflammation.

Abstract TMEM-040: TUMOR ASSOCIATED MACROPHAGES MEDIATES THE ONCOGENIC EFFECT OF FGF18 IN OVARIAN CANCER

Abstract Epithelial ovarian cancer is the fifth leading cause of cancer-related death among women and has the highest case-fatality rate among gynecologic cancers. Effective management of this deadly disease still remains scanty due to the inadequate understanding of the molecular mechanisms associated with the tumor progression and prognosis. We have previously reported the tumor-promoting role of Fibroblast growth factor 18 (FGF18), which have been identified through genomic analysis of ovarian tumors. Initial studies suggested FGF18 promotes ovarian tumor progression through NF-κB pathway mediated elevation of proinflammatory cytokines, which mediate the increased tumor burden, angiogenesis and infiltration of macrophages in FGF18 expressing xenografts. Based on these findings, the present study aims to delineate the mechanism by which FGF18 modulates the phenotype of tumor associated macrophages (TAMs) as well as the reciprocal tumor-promoting feedback from the TAMs which potentially contributes FGF18 mediated ovarian tumorigenesis. Immunohistochemical staining of a tissue array comprising 216 archived high-grade advanced stage serous ovarian tumor specimens revealed a significant correlation between the FGF18 expression and infiltration of M2-polarized macrophages (by CD163). In addition, the density of intratumoral M2 TAMs inversely associated with patients' overall survival as a negative prognostic factor independent to age, tumor grade and debulking status. These findings suggest intratumoral TAMs may play an important role in ovarian tumorigenesis. In vitro co-culture studies further demonstrated that human monocyte cell line THP-1 or murine peritoneal macrophage cell line IC-21 could significantly activate the NF-κB pathway and increase the production of various proinflammatory cytokines in ovarian cancer cells. Co-culture also increased the migratory, adhesive and angiogenic potential of ovarian cancer cells cells. Addition of an IKKβ inhibitor into the co-culture system nullified the cytokine production and oncogenic changes of tumor cells promoted by monocytes/macrophages, implicating the involvement of NF-κB signaling in the crosstalk. Conversely, liposome clodronate mediated in vivo macrophage depletion significantly shrunk the tumor burden of the intraperitoneal xenograft derived from FGF18 overexpressing SKOV3 cells, while minimal effect was observed over the control RFP overexpressing xenograft. Besides, macrophage depletion also markedly decreased the intratumoral microvessel density induced by FGF18 overexpression. Taken together, our data potentially suggest TAMs may mediate the FGF18 related ovarian tumorigenesis by augmenting the proinflammatory status of the tumors. Citation Format: Wei Wei and Michael J. Birrer. TUMOR ASSOCIATED MACROPHAGES MEDIATES THE ONCOGENIC EFFECT OF FGF18 IN OVARIAN CANCER [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr TMEM-040.