THP-1 Differentiation Research Articles

Brassinin, a brassica-derived phytochemical, regulates monocyte-to-macrophage differentiation and inflammatory responses in human monocytes and murine macrophages.

The effects and molecular mechanisms of brassinin (BR), an indole phytoalexin from cruciferous vegetables, on monocyte-to-macrophage differentiation and inflammatory responses were investigated in this study. Inflammatory responses from RAW264.7 cells and THP-1 were stimulated by lipopolysaccharide (1µg/ml), and monocyte-to-macrophage differentiation of THP-1 was induced by phorbol myristate acetate (50ng/ml). The production of inflammatory mediators was determined by ELISA, Western blot or real-time PCR. Reactive oxygen species were examined by DCFH-DA assay. Brassinin at 50µm suppressed lipopolysaccharide-induced production of nitric oxide synthase, cyclooxygenase-2, prostaglandin E2 and reactive oxygen species by 90%, 69%, 52% and 41%, respectively, in RAW264.7 cells. In THP-1 cells, BR inhibited phorbol myristate acetate-induced monocyte-to-macrophage differentiation by suppressing cluster of differentiation molecule β and CD36. In addition, BR suppressed translocation of nuclear factor 'kappa-light-chain-enhancer' of activated B cells (NF-κB) into the nucleus. However, BR activated the nuclear factor erythroid-derived 2-like 2 (Nrf2) and its target molecules hemoxygenase-1 (HO-1) and NAD(P)H: quinone oxidoreductase 1 (NQO1), with an increase in nuclear translocation of Nrf2. Brassinin suppressed monocyte-to-macrophage differentiation and inflammatory responses by differentially regulating Nrf2 and NF-κB signallings.

In Vitro Anti-Inflammatory and Immunomodulatory Activities of an Extract from the Roots of Bupleurum rotundifolium

Background: Some Bupleurum species, such as the Bupleurum chinense DC. or the Bupleurum scorzonerifolium Willd have been extensively studied (especially their roots) for the treatment of inflammation. In contrast, only compounds extracted from the aerial parts of Bupleurum rotundifolium have been studied and showed anti-inflammatory or antiproliferative activities. This study was conducted to investigate the antioxidant, anti-inflammatory, and immunomodulatory effects of Bupleurum rotundifolium roots. Methods: To tackle the various aspects of inflammation, we studied in vitro a methanolic extract from the roots of Bupleurum rotundifolium on peripheral blood mononuclear cells (PBMCs), polymorphonuclear neutrophils (PMNs), and the monocytic cells THP-1. Its antioxidant capacities and iron-chelating activity were assessed. The extract was tested on THP-1 differentiation, reactive oxygen species (ROS) production by leukocytes, neutrophils chemotaxis, cytokines, PGE2 production, and NF-κB activation in PBMCs. Results: The extract showed a decreased ROS production in stimulated cells. It increased PBMC chemokine secretion and up-regulated the differentiation of THP-1 monocytes into macrophage-like cells, indicating a potential interest of the extract in the resolution of acute inflammation. In addition, the analysis of cytokine production suggests that Bupleurum rotundifolium has immunomodulatory properties. Conclusions: Cytokines secretion, especially IL-1β and IL-12p70, provided us with a set of indicators suggesting that the extract might be able to drive the polarization of macrophages and lymphocytes toward a Th2 anti-inflammatory profile in excessive inflammation.

Nemorosone inhibits the proliferation and migration of hepatocellular carcinoma cells

AimsIn the tumor microenvironment, dysregulated immune cells could promote tumor progression, invasion and metastasis, by establishing a symbiotic relationship with cancer cells. A pivotal role is played by monocyte recruitment and induction of tumor-associated macrophages (TAMs), which provide immunosuppression and tumorigenesis. The effect of nemorosone, an antiproliferative phytocomponent present in Cuban Propolis, on TAM-induced tumor progression remains to be elucidated. Here we investigated the symbiotic relationship between monocytic leukemia THP-1 and hepatocellular carcinoma HepG2 cells, and the role of nemorosone in preventing TAM-induced tumor growth. Main methodsMacrophage differentiation induced by HepG2-conditioned medium was assessed by flow cytometry, analysis of secreted molecules and cytokine expression. The effect of nemorosone and/or conditioned THP-1-medium on HepG2 proliferation was evaluated by MTT assay, colony formation, cells cycle and migration assays. Key findingsHepG2 cells induced THP-1 recruitment and differentiation to macrophages. When compared with control THP-1 cells, differentiated THP-1 showed a significant increase of the matrix metalloproteinases MMP-2 and MMP-9 expression (P < 0.01), and slightly induced HepG2 cells growth. This effect was counteracted by nemorosone, which also significantly inhibited colony formation (P < 0.01) and migratory capacity of HepG2 cells, driving a high percentage of cells (80%) to the G0/G1 phase. SignificanceHepG2-conditioned medium is a suitable model for THP-1 modulation and differentiation. Moreover, nemorosone significantly inhibits the proliferation of HepG2 cells, both in presence and absence of the soluble factors secreted by TAMs. Further studies are needed to elucidate the role of this natural compound in the HCC-TAM relationship.

Differential expression of the TPα and TPβ isoforms of the human T Prostanoid receptor during chronic inflammation of the prostate: Role for FOXP1 in the transcriptional regulation of TPβ during monocyte-macrophage differentiation

Inflammation is linked to prostate cancer (PCa) and to other diseases of the prostate. The prostanoid thromboxane (TX)A2 is a pro-inflammatory mediator implicated in several prostatic diseases, including PCa. TXA2 signals through the TPα and TPβ isoforms of the T Prostanoid receptor (TP) which exhibit several functional differences and transcriptionally regulated by distinct promoters Prm1 and Prm3, respectively, within the TBXA2R gene. This study examined the expression of TPα and TPβ in inflammatory infiltrates within human prostate tissue. Strikingly, TPβ expression was detected in 94% of infiltrates, including in B- and T-lymphocytes and macrophages. In contrast, TPα was more variably expressed and, where present, expression was mainly confined to macrophages. To gain molecular insight into these findings, expression of TPα and TPβ was evaluated as a function of monocyte-to-macrophage differentiation in THP-1 cells. Expression of both TPα and TPβ was upregulated following phorbol-12-myristate-13-acetate (PMA)-induced differentiation of monocytic THP-1 to their macrophage lineage. Furthermore, FOXP1, an essential transcriptional regulator down-regulated during monocyte-to-macrophage differentiation, was identified as a key trans-acting factor regulating TPβ expression through Prm3 in THP-1 cells. Knockdown of FOXP1 increased TPβ, but not TPα, expression in THP-1 cells, while genetic reporter and chromatin immunoprecipitation (ChIP) analyses established that FOXP1 exerts its repressive effect on TPβ through binding to four cis-elements within Prm3. Collectively, FOXP1 functions as a transcriptional repressor of TPβ in monocytes. This repression is lifted in differentiated macrophages, allowing for upregulation of TPβ expression and possibly accounting for the prominent expression of TPβ in prostate tissue-resident macrophages.

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.

4-Phenylbutyrate protects against atherosclerotic lesion growth by increasing the expression of HSP25 in macrophages and in the circulation of Apoe-/- mice.

Endoplasmic reticulum stress plays an important role in cardiovascular disease (CVD) and atherosclerosis. We aimed to assess the ability of 4-phenylbutyrate (4-PBA), a small chemical chaperone administered via drinking water, to reduce atherosclerotic lesion size in chow-fed apolipoprotein (Apo) e-/- mice and to identify mechanisms that contribute to its antiatherogenic effect. Chow-fed 17-wk-old female Apoe-/- mice treated with 4-PBA-supplemented drinking water for 5 wk exhibited smaller lesions as well as increased plasma levels of heat shock protein (HSP) 25, the mouse homolog of human HSP27, compared with controls. In addition, 4-PBA inhibited cell death and increased HSP27 expression as measured by real-time PCR and immunoblotting, as well as induced nuclear localization of its transcription factor, heat shock factor 1, in human monocyte/macrophage (THP-1) cells. Furthermore, HSP27 small interfering RNA diminished the protective effect of 4-PBA on THP-1 macrophage attachment and differentiation. In summary, drinking water containing 4-PBA attenuated early lesion growth in Apoe-/- mice fed a chow diet and increased expression of HSP25 and HSP27 in macrophages and HSP25 in the circulation of Apoe-/- mice. Given that increased expression of HSP27 is inversely correlated with CVD risk, our findings suggest that 4-PBA protects against the early stages of atherogenesis in part by enhancing HSP27 levels, leading to inhibition of both macrophage cell death and monocyte-macrophage differentiation.-Lynn, E. G., Lhoták, Š., Lebeau, P., Byun, J. H., Chen, J., Platko, K., Shi, C., O'Brien, E. R., Austin, R. C. 4-Phenylbutyrate protects against atherosclerotic lesion growth by increasing the expression of HSP25 in macrophages and in the circulation of Apoe-/- mice.

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.

Pardaxin Promoted Differentiation and Maturation of Leukemic Cells via Regulating TLR2/MyD88 Signal against Cell Proliferation.

Objective Leukemia is a cancer of the blood cells. Leukemic THP-1 and U937 cells were used in this study as monocytic effectors cells for proliferation responses and macrophage-like cells induction in leukemia. Pardaxin is an antimicrobial peptide isolated from the marine fish species. Methods After treatment for 5 days, pardaxin significantly suppressed cell viability and arrested cell cycle at G0/G1 phase in leukemic cells which were evaluated. Results Pardaxin also induced cell differentiation and maturation of THP-1 and U937 cells into macrophage-like cells with phagocytotic ability. Moreover, pardaxin elevated the expression of MyD88 but not toll-like receptor (TLR)-2 in both leukemic cells. TLR-2 blocking peptide was used to confirm that pardaxin attenuated phagocytotic ability and superoxide anion production in leukemic cells via activating MyD88 protein. Conclusions These findings suggested that pardaxin has a therapeutic potential for leukemia.

996 Atopic dermatitis model on RHE in presence of S.aureus and infiltrating immuno-competent cells

Atopic dermatitis (AD) is a common skin disease linked to a dysregulation of the immune system and an impaired epidermal barrier. AD lesions show an increased susceptibility to infection by S. aureus, which triggers the production of thymic stromal lymphopoietin (TSLP) by keratinocytes. Based on Reconstructed Human Epidermis (RHE) with a double porosity polycarbonate filter allowing THP-1 monocyte infiltration, a unique in vitro model has been developed to better investigate the cutaneous immune response activated by S. aureus in AD. RHE with impaired barrier were colonized with S. aureus at 2x106 CFU/insert. THP-1 infiltrated the RHE for 4 hours. Tissue response was assessed after 4 hours (atopic rash), 16 and 48 hours (delayed inflammation) by qRT-PCR of inflammatory (TNF-α, TSLP, TLR-2), antimicrobial (human β-defensin 2) response and barrier (filaggrin, CLDN1, ZO-1) markers. Markers of THP-1 differentiation in APC were evaluated (CD86, CD14 and CD11b) as well as Th1/Th2-associated cytokines (TNF-α, IFN-γ, IL-4, IL-5, IL-8, IL-10, IL-13). For selected biomarkers protein expression was confirmed by immunolabelling. THP-1 successfully infiltrated the colonized RHE and differentiated towards a dendritic phenotype. A significant increase of the expression of IL-8, TLR-2 and TNF-α indicated an inflammation in the THP-1-RHE model in presence of S. aureus. TSLP expression presented a 20-fold increase while filaggrin expression decreased when THP-1 were in direct contact with the keratinocytes. S. aureus colonization of the RHE co-cultured with THP-1 also decreased filaggrin expression. Human β-defensin 2 increased by more than 100-fold in the AD model, reflecting keratinocyte reaction to S. aureus. The THP-1-RHE cell migration model seems to fully recapitulate the features of AD in vitro by taking into account the keratinocyte innate and inflammatory response and the immune-mediated response in presence of a S. aureus stable colonization.

Inhibition of SOX9 Promotes Inflammatory and Immune Responses of Dental Pulp

IntroductionThe process of pulpitis is characterized by extracellular matrix imbalance and inflammatory cell infiltration. As an essential transcription factor, sex-determining region Y-box 9 (SOX9) is significantly inhibited by tumor necrosis factor alpha in inflammatory joint diseases. The aim of this study was to explore the role of SOX9 in extracellular matrix balance, cytokine expression, and the immune response in dental pulp. MethodsThe expression of SOX9 in normal and inflamed pulp tissue/human dental pulp cells (HDPCs) was detected by immunohistochemistry, Western blot, and quantitative polymerase chain reaction (qPCR). SOX9 small interfering RNA was used to knock down SOX9 expression of dental cells in vitro; extracellular matrix imbalance was analyzed by qPCR, Western blot, and gelatin/collagen zymography, and the secretion of cytokines was scanned by antibody arrays. The immune response of THP-1 was investigated by cell migration assay, cell attachment assay, phagocytosis assay, and enzyme-linked immunosorbent assay. The interaction of SOX9 with target genes was explored by chromatin immunoprecipitation (ChIP). ResultsSOX9 was strongly expressed in normal dental pulp tissue and HDPCs and reduced in inflamed pulp. SOX9 knockdown could inhibit the production of type I collagen, stimulate the enzymatic activities of MMP2 and MMP13, and regulate the production of interleukin (IL) 8 of HDPCs. SOX9 knockdown also effectively suppressed the differentiation and functional activities of THP-1. ChIP showed that the binding of the SOX9 protein with matrix metalloproteinase (MMP)-1, MMP-13, and IL-8 gene promoters was reduced after being treated with recombinant human tumor necrosis factor alpha. ConclusionsSOX9 was inhibited in inflamed dental pulp and may participate in the regulation of extracellular matrix balance, the inflammatory process, and the immune response.

AURKA Suppresses Leukemic THP-1 Cell Differentiation through Inhibition of the KDM6B Pathway.

Aberrations in histone modifications are being studied in mixed-lineage leukemia (MLL)-AF9-driven acute myeloid leukemia (AML). In this study, we focused on the regulation of the differentiation of the MLL-AF9 type AML cell line THP-1. We observed that, upon phorbol 12-myristate 13-acetate (PMA) treatment, THP-1 cells differentiated into monocytes by down-regulating Aurora kinase A (AURKA), resulting in a reduction in H3S10 phosphorylation. We revealed that the AURKA inhibitor alisertib accelerates the expression of the H3K27 demethylase KDM6B, thereby dissociating AURKA and YY1 from the KDM6B promoter region. Using Flow cytometry, we found that alisertib induces THP-1 differentiation into monocytes. Furthermore, we found that treatment with the KDM6B inhibitor GSK-J4 perturbed the PMA-mediated differentiation of THP-1 cells. Thus, we discovered the mechanism of AURKA–KDM6B signaling that controls the differentiation of THP-1 cells, which has implications for biotherapy for leukemia.

Immunomodulatory effect of structurally-characterized mushroom sclerotial polysaccharides isolated from Polyporus rhinocerus on human monoctyes THP-1

Two high molecular weight (MW larger than 788 kDa) mushroom polysaccharides extracted by hot water (PRW) and micronized sonication-assisted cold alkali (mPRSon), were isolated from the sclerotia of Polyporus rhinocerus. PRW was a polysaccharide-protein complex with a highly branched (degree of branching DB 0.60) β-D-mannoglucan while mPRSon was a hyper-branched β-glucan (DB 0.85). PRW could bind to complement receptor 3 (CR3) and significantly induced THP-1 differentiation evidenced by cell morphology and enhanced phagocytic activity. A significant increase in the expression of surface markers including CD14, CD11b and HLA-DR as well as enhanced production of cytokines including IL-8, RANTES, GRO, MCP-1, MCP-2 and IL-6 were observed on THP-1 cells treated with PRW. In contrast, mPRSon could not either activate the Dectin-1 signaling or induce the THP-1 differentiation. High MW and DB mushroom sclerotial polysaccharide which has a structure of heteroglycan and protein is more immunopotent than pure homoglycan.

Novel click modifiable thioquinazolinones as anti-inflammatory agents: Design, synthesis, biological evaluation and docking study

Click chemistry was used to synthesize a new series of thioquinazolinone molecules equipped with propargyl moiety,1,2,3-triazolyl and isoxazolyl rings. Our design was based on merging pharmacophores previously reported to exhibit COX-2 inhibitory activities to a thioquinazolinone-privileged scaffold. The synthesized compounds were subjected to in vitro cyclooxygenase COX-1/COX-2 and 15-LOX inhibition assays. Compounds 2c, 3b, 3h, 3j, and 3k showed COX-2 inhibition with IC50 (μM) 0.18, 0.19, 0.11, 0.16 and 0.17 respectively. These values were compared to celecoxib (IC50 0.05 μM), diclofenac (IC50 0.8 μM) and indomethacin (IC50 0.49 μM) reference drugs. They also showed 15-LOX inhibition with IC50 (μM) 6.21, 4.33, 7.62, 5.21 and 3.98 respectively. These values were compared with Zileuton (IC50 2.41 μM) and Meclofenamate sodium (IC50 5.64 μM) as positive controls. These compounds were further challenged by PMA-induced THP-1 differentiation assay where compounds 2c and 3j inhibited monocyte to macrophage differentiation efficiently with IC50 values of 4.78 μM and 5.63 μM, respectively, compared to that of diclofenac sodium (4.86 μM). On the other hand, 3h demonstrated a significantly increased potency compared to diclofenac in this assay (IC50 = 0.13 μM). The same compounds exhibited significant in vivo anti-inflammatory effect as indicated by the formalin-induced rat-paw edema test. Docking experiments of compounds 2c, 3b, 3h, 3j, and 3k into COX-2 binding pocket have been conducted, where strong binding interactions have been identified and effective overall docking scores have been recorded. Their drug-likeness has been assessed using Molinspiration, Molsoft and Pre-ADMET software products.

Deregulated MicroRNA-21 Expression in Monocytes from HIV-Infected Patients Contributes to Elevated IP-10 Secretion in HIV Infection.

Persistent activation and inflammation impair immune response and trigger disease progression in HIV infection. Emerging evidence supports the supposition that excessive production of interferon-inducible protein 10 (IP-10), a critical inflammatory cytokine, leads to immune dysfunction and disease progression in HIV infection. In this study, we sought to elucidate the cause of the upregulated production of IP-10 in HIV infection and explore the underlying mechanisms. Bolstering miR-21 levels using mimics resulted in the obvious suppression of lipopolysaccharide (LPS)-induced IP-10 in monocyte leukemia cells THP-1 and vice versa. The analysis of the primary monocytes of HIV patients revealed significantly less miR-21 than in healthy controls; this was opposite to the tendency of IP-10 levels in plasma. The secretion of IP-10 due to LPS stimulation was not affected by miR-21 modulation in the differentiated THP-1 macrophages (THP-1-MA). We found a novel switch, IFN-stimulated gene 15 (ISG15), which triggers the expression of IP-10 and is significantly upregulated during the differentiation of THP-1 into THP-1-MA. The inhibition of ISG15 can restore the regulation of IP-10 by miR-21. In summary, IP-10 expression in monocytes is regulated by miR-21, whereas in macrophages, this fine-tuning is attenuated by the enhanced expression of ISG15. This study paves the way to a comprehensive understanding of the molecular regulatory mechanism of IP-10, a key point in immune intervention strategy.

Abstract 3088: Combination of 4SC-202 and IFN-γ restores mature APC phenotype in AML cells

Abstract Acute myeloid leukemia (AML) is characterized by a differentiation block resulting in accumulation of immature myeloid cells. Differentiation therapy, e.g. with all-trans retinoic acid (ATRA) has become a part of standard treatment for the acute promyelocytic leukemia variant of AML. Other agents like cytokines and HDAC inhibitors were shown to enhance differentiation of AML cells, which results in apoptosis induction or re-sensitization to ATRA. However, a differentiation therapy resulting in the restoration of antigen presenting function of AML cells would be a more desirable asset, since such cells will be able to induce or enhance immune response against residual malignant AML cells. Here, we analyzed the impact of 4SC-202 alone and in combination with different cytokines on differentiation of AML cell lines THP-1, HL-60 and MOLM-13 (AML-M4, -M2, and -M5 class, respectively). Myeloid lineage markers CD11b, CD86, CD14 and HLA-DR were analyzed by flow cytometry and quantitative PCR. Cytokine production was measured using Luminex multiplex system. Furthermore, chromatin immunoprecipitation (ChIP) was performed to assess the direct impact of 4SC-202 on promoter histone acetylation of differentiation genes. 4SC-202 is an orally available clinical stage epigenetic small molecule inhibitor which specifically targets histone deacetylases HDAC class I isoenzymes 1-3 as well as the lysine-specific demethylase LSD1 (KDM1A). 4SC-202 treatment of AML cell lines resulted in a dose-dependent increase of myeloid differentiation genes CD86 and ITGAM (=CD11b) on mRNA and protein level. On the chromatin level, 4SC-202 correspondently induced an upregulation of chromatin activation marks like acetylation of histone H3 lysine 9 and 27 (H3K9ac, H3K27ac) at the promoters of these genes. Interestingly, reference compounds like ATRA, bexarotene, GM-CSF and TNF-α were not as efficacious as 4SC-202 in the induction of differentiation markers in AML cells, whereas IFN-γ strongly induced MHC class II molecules HLA-DR and myeloid lineage marker CD14. Finally, the combination of 4SC-202 and IFN-γ resulted in a strong and complementary induction of differentiation and APC markers CD11b, CD14, CD86 and HLA-DR resulting in a CD86high/HLA-DRhigh phenotype which was able to produce pro-inflammatory cytokines. In summary, we demonstrate that 4SC-202 induces differentiation of AML cells of different subtypes and thus provide promising preclinical data for applying this novel epigenetic modulator for differentiation therapy of AML. Moreover, the mature APC phenotype induced by the combination of 4SC-202 and IFN-γ may further suggest an application in consolidation treatment of AML to enhance or even elicit a tumor-specific T-cell response against residual malignant AML cells preventing AML recurrence. Citation Format: Anne Catherine Bretz, Ulrike Parnitzke, Kerstin Kronthaler, Svetlana Hamm. Combination of 4SC-202 and IFN-γ restores mature APC phenotype in AML cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3088. doi:10.1158/1538-7445.AM2017-3088

Epithelial Cells Secrete Interferon-γ Which Suppresses Expression of Receptor Activator of Nuclear Factor Kappa-B Ligand in Human Mandibular Osteoblast-Like Cells.

Prostaglandin (PG)E2 accumulates in inflamed periodontal tissue and induces receptor activator of nuclear factor kappa-B ligand (RANKL)-RANK-osteoprotegerin (OPG) signaling associated with bone resorption. Although oral epithelial cells maintain tissue homeostasis, the role of these cells in RANKL regulation remains unknown. To mimic an inflamed condition, RANKL upregulation in human mandibular osteoblast-like cells (HMOBs) were stimulated with PGE2. Effect of recombinant human interferon (IFN)-γ or epithelial-derived IFN-γ in constitutively released or Porphyromonas gingivalis lipopolysaccharide (PgLPS)-stimulated epithelial supernatant was investigated in HMOBs. Some HMOBs were pretreated with an anti-IFN-γ antibody before PGE2 stimulation. THP-1 human monocytes and HMOBs were cocultured in a transwell system to investigate RANKL-driven THP-1 osteoclastic activity. PGE2 significantly increased RANKL messenger RNA (mRNA) and protein in HMOBs in a dose-dependent manner, while OPG protein remained similar to baseline. Epithelial cells constitutively released IFN-γ, which was substantially increased by PgLPS. HMOBs treated with epithelial supernatant or recombinant IFN-γ, concurrently with PGE2 stimulation, reduced RANKL, but not OPG, expression. In contrast, anti-IFN-γ antibody reversed the effect of epithelial mediators on RANKL expression. When cocultured with THP-1, RANKL released by PGE2-stimulated HMOBs is adequate to drive THP-1 differentiation as osteoclastogenic gene expression and bone resorption pit are increased. However, recombinant IFN-γ, or IFN-γ derived from oral epithelial cells, suppressed RANKL expression at both the mRNA and protein level, resulting in decreased THP-1-derived osteoclastic activity. Oral epithelial cells interact with HMOBs by releasing IFN-γ to regulate RANKL expression and contribute to osteoclastogenesis.

Glycosylation Changes Triggered by the Differentiation of Monocytic THP-1 Cell Line into Macrophages

The human acute monocytic leukemia cell line THP-1 is widely used as an in vitro phagocytic cell model because it exhibits several immune properties similar to native monocyte-derived macrophages. In this study, we investigated the alteration of N- and O-linked glycans as well as glycosphingolipids, during THP-1 differentiation, combining mass spectrometry, flow cytometry, and quantitative real-time PCR. Mass spectrometry revealed that macrophage differentiation led to a marked upregulation of expression of GM3 ganglioside as well as an increase in complex-type structures, particularly triantennary glycans, occurring at the expense of high-mannose N-glycans. Moreover, we observed a slight decrease in the proportion of multifucosylated N-glycans and α2,6-sialylation. The uncovered changes in glycosylation correlated with variations of gene expression of relevant glycosyltransferases and glycosidases including sialyltransferases, β-N-acetylglucosaminyltransferases, fucosyltransferases, and neuraminidase. Furthermore, using flow cytometry and antibodies directed against glycan structures, we confirmed that the alteration of glycosylation occurs at the cell surface of THP-1 macrophage-like cells. Altogether, we established that macrophagic maturation of THP-1 induces dramatic modifications of the surface glycosylation pattern that may result in differential interaction of monocytic and macrophagic THP-1 with immune or bacterial lectins.

Intracellular NAD+ levels are associated with LPS-induced TNF-α release in pro-inflammatory macrophages.

Metabolism and immune responses have been shown to be closely linked and as our understanding increases, so do the intricacies of the level of linkage. NAD(+) has previously been shown to regulate tumour necrosis factor-α (TNF-α) synthesis and TNF-α has been shown to regulate NAD(+) homoeostasis providing a link between a pro-inflammatory response and redox status. In the present study, we have used THP-1 differentiation into pro- (M1-like) and anti- (M2-like) inflammatory macrophage subset models to investigate this link further. Pro- and anti-inflammatory macrophages showed different resting NAD(+) levels and expression levels of NAD(+) homoeostasis enzymes. Challenge with bacterial lipopolysaccharide, a pro-inflammatory stimulus for macrophages, caused a large, biphasic and transient increase in NAD(+) levels in pro- but not anti-inflammatory macrophages that were correlated with TNF-α release and inhibition of certain NAD(+) synthesis pathways blocked TNF-α release. Lipopolysaccharide stimulation also caused changes in mRNA levels of some NAD(+) homoeostasis enzymes in M1-like cells. Surprisingly, despite M2-like cells not releasing TNF-α or changing NAD(+) levels in response to lipopolysaccharide, they showed similar mRNA changes compared with M1-like cells. These data further strengthen the link between pro-inflammatory responses in macrophages and NAD(+). The agonist-induced rise in NAD(+) shows striking parallels to well-known second messengers and raises the possibility that NAD(+) is acting in a similar manner in this model.

Pathways related to PMA-differentiated THP1 human monocytic leukemia cells revealed by RNA-Seq.

Previous analyses have reported that the human monocytic cell line THP1 can be differentiated into cells with macrophage-like characteristics by phorbol 12-myristate 13-acetate (PMA). However, little is known about the mechanism responsible for regulating this differentiation process. Here, we performed high-throughput RNA-Seq analysis to investigate the genes differently expressed in THP1 cells treated with and without PMA and examined those that may be responsible for the PMA-induced differentiation of monocytes into macrophages. We found 3,000 genes to be differentially expressed after PMA treatment. Gene ontology analysis revealed that genes related to cellular processes and regulation of biological processes were significantly enriched. KEGG analysis also demonstrated that the differentially expressed genes (DEGs) were significantly enriched in the PI3K/AKT signaling pathway and phagosome pathway. Importantly, we reveal an important role of the PI3K/AKT pathway in PMA-induced THP1 cell differentiation. The identified DEGs and pathways may facilitate further study of the detailed molecular mechanisms of THP1 differentiation. Thus, our results provide numerous potential therapeutic targets for modulation of the differentiation of this disease.