Human Monocytic Leukemia THP-1 Cells Research Articles

Nucleophosmin Mutations Induce Chemosensitivity in THP-1 Leukemia Cells by Suppressing NF-κB Activity and Regulating Bax/Bcl-2 Expression.

Nucleophosmin (NPM1) - a gene that encodes for a nuclear protein with multiple functions. Mutations in NPM1 are seen in approximately one-third of acute myeloid leukemia (AML) and are generally associated with good response to induction chemotherapy. However, the mechanisms underlying this chemosensitivity are still unknown. Recent studies have established that nuclear factor-κB (NF-κB) activation is a key response of leukemia cell to chemotherapy. In this study, we transfected human monocytic leukemia THP-1 cells with the vector expressing NPM1 mutation variant (NPM1mA), and confirmed overexpression of NPM1mA at mRNA and protein levels by reverse transcription PCR (RT-PCR) and immunohistochemistry, respectively. The effects of NPM1 mutations on chemotherapeutical agents induced apoptosis, NF-κB activity and gene expression were examined using flow cytometry, luciferase reporter assays, quantitative real time PCR (qRT-PCR) and Western blot. We found that overexpression of NPM1mA in THP-1 cells sensitized these cells to apoptosis induced by chemotherapeutical agents such as daunorubicin (DNR) and cytarabine (Ara-C). Moreover, we demonstrated that expression of NPM1 mA reduced the NF-κB transcription activity of THP-1 cells upon drug treatment. In addition, restoration of NF-κB activity via TNF-α stimulation could attenuate the effect of NPM1mA overexpression on DNR-and Ara-C-induced apoptosis. Interestingly, expression of NPM1mA could upregulate Bax and downregulate Bcl-2 at mRNA and protein levels in THP-1 cells when treated with DNR or Ara-C. We also demonstrated that restoration of NF-κB activity via TNF-α pre-treatment reversed the effect of NPM1mA on the Bax/Bcl-2 expression. Furthermore, evaluation of gene expression data from The Cancer Genome Atlas (TCGA) dataset revealed that NPM1-mutated patients showed a higher expression of Bax and a lower expression of Bcl-2. These results suggest that the NPM1 gene mutations could confer increased sensitivity to chemotherapeutic agents, at least in part, by suppressing NF-κB activity and regulating Bax/Bcl-2 expression.

Inhibitory effects of Kaempferia parviflora extract on monocyte adhesion and cellular reactive oxygen species production in human umbilical vein endothelial cells.

The rhizome of Kaempferia parviflora (KP) is used in traditional Thai medicine. In this study, we investigated the effects of an ethanol KP extract and two of its components [5,7-dimethoxyflavone (DMF) and 5-hydroxy-3,7,3',4'-tetramethoxyflavone (TMF)] on monocyte adhesion and cellular reactive oxygen species (ROS) production in human umbilical vein endothelial cells (HUVECs), which provide an in vitro model of events relevant to the development and progression of atherosclerosis. RAW264.7 mouse macrophage-like cells were incubated with various concentrations of KP extract or polymethoxyflavonoids and stimulated with lipopolysaccharide prior to measuring nitrite levels in the culture media. Monocyte adhesion was evaluated by measuring the fluorescently labeled human monocytic leukemia THP-1 cells that is attached to tumor necrosis factor-α (TNF-α)-stimulated HUVECs. Cellular ROS production was assessed by measuring cellular antioxidant activity using pyocyanin-stimulated HUVECs. KP extract and DMF reduced nitrite levels (as indicator of nitric oxide production) in LPS-stimulated RAW264.7 cells and also inhibited THP-1 cell adhesion to HUVECs. These treatments induced mRNA expression of endothelial nitric oxide synthase in TNF-α-stimulated HUVECs and downregulated that of various cell adhesion molecules, inflammatory mediators, and endothelial function-related genes. Angiotensin-converting enzyme activity was inhibited by KP extract in vitro. Furthermore, KP extract, DMF, and TMF inhibited the production of cellular ROS in pyocyanin-stimulated HUVECs. KP extract, DMF, and TMF showed potential anti-inflammatory and antioxidant effects in these in vitro models, properties that would inhibit the development and progression of atherosclerosis.

Suppression of NRF2–ARE activity sensitizes chemotherapeutic agent-induced cytotoxicity in human acute monocytic leukemia cells

Nuclear factor erythroid 2-related factor 2 (NRF2), a master regulator of the antioxidant response element (ARE)-dependent transcription, plays a pivotal role in chemical detoxification in normal and tumor cells. Consistent with previous findings that NRF2–ARE contributes to chemotherapeutic resistance of cancer cells, we found that stable knockdown of NRF2 by lentiviral shRNA in human acute monocytic leukemia (AML) THP-1 cells enhanced the cytotoxicity of several chemotherapeutic agents, including arsenic trioxide (As2O3), etoposide and doxorubicin. Using an ARE-luciferase reporter expressed in several human and mouse cells, we identified a set of compounds, including isonicotinic acid amides, isoniazid and ethionamide, that inhibited NRF2–ARE activity. Treatment of THP-1 cells with ethionamide, for instance, significantly reduced mRNA expression of multiple ARE-driven genes under either basal or As2O3-challenged conditions. As determined by cell viability and cell cycle, suppression of NRF2–ARE by ethionamide also significantly enhanced susceptibility of THP-1 and U937 cells to As2O3-induced cytotoxicity. In THP-1 cells, the sensitizing effect of ethionamide on As2O3-induced cytotoxicity was highly dependent on NRF2. To our knowledge, the present study is the first to demonstrate that ethionamide suppresses NRF2–ARE signaling and disrupts the transcriptional network of the antioxidant response in AML cells, leading to sensitization to chemotherapeutic agents.

Picfeltarraenin IA inhibits lipopolysaccharide-induced inflammatory cytokine production by the nuclear factor-κB pathway in human pulmonary epithelial A549 cells.

The present study aimed to investigate the effect of picfeltarraenin IA (IA) on respiratory inflammation by analyzing its effect on interleukin (IL)-8 and prostaglandin E2 (PGE2) production. The expression of cyclooxygenase 2 (COX2) in human pulmonary adenocarcinoma epithelial A549 cells in culture was also examined. Human pulmonary epithelial A549 cells and the human monocytic leukemia THP-1 cell line were used in the current study. Cell viability was measured using a methylthiazol tetrazolium assay. The production of IL-8 and PGE2 was investigated using an enzyme-linked immunosorbent assay. The expression of COX2 and nuclear factor-κB (NF-κB)-p65 was examined using western blot analysis. Treatment with lipopolysaccharide (LPS; 10 µg/ml) resulted in the increased production of IL-8 and PGE2, and the increased expression of COX2 in the A549 cells. Furthermore, IA (0.1-10 µmol/l) significantly inhibited PGE2 production and COX2 expression in cells with LPS-induced IL-8, in a concentration-dependent manner. The results suggested that IA downregulates LPS-induced COX2 expression, and inhibits IL-8 and PGE2 production in pulmonary epithelial cells. Additionally, IA was observed to suppress the expression of COX2 in THP-1 cells, and also to regulate the expression of COX2 via the NF-κB pathway in the A549 cells, but not in the THP-1 cells. These results indicate that IA regulates LPS-induced cytokine release in A549 cells via the NF-κB pathway.

Alda-1 Constrains TLR-Induced Cytokine Overproduction By Fanconi Anemia Macrophages By Enhancing ALDH1A1 Activity

Replication and survival of FA-deficient hematopoietic stem and progenitor cells (HSPC) are highly suppressed by exposure to inflammatory cytokines including TNFα, IL-1β, IFNγ, and MIP-1α. Additionally, FA macrophages exposed to specific toll-like receptor (TLR) ligands overproduce the very inflammatory cytokines that exhaust the stem cell pool. Recent evidence using double knockout mice suggests that aldehyde dehydrogenases (ALDH) may play a role in protecting FA HSPC but the mechanisms involved are unclear. We tested the hypothesis that the TLR-dependent overproduction of inflammatory cytokines in FA macrophages results from a loss of FA protein-dependent ALDH function. Control (T-shNT) or FANCC-deficient (T-shFC) THP-1 human monocytic leukemia cells were treated with Alda-1 (a small molecule ALDH agonist known to enhance the activity of both ALDH1A1 and ALDH2), before exposing them to the TLR-7/8 agonist R848 and quantifying the production of TNFα (ELISA of 24- culture supernatants). While R848 alone induced a 6-fold increase in TNFα production by the FANCC-deficient cells, Alda-1 suppressed TLR-induced TNFα production in a dose-dependent manner, by both T-shNT and T-shFC cells (Fig. 1). To identify the ALDH isoform that played a role in the suppression of TNFα production, we ectopically expressed ALDH1A1 or ALDH2 in T-shNT and T-shFC cells. Overexpression of ALDH2 had little effect on R848-induced TNFα production, but ALDH1A1 overexpression completely suppressed TNFα production by FANCC-deficient cells and suppressed (by approximately 50%) the production of TNFα by control cells (Fig. 2). Likewise, in loss-of-function studies, siRNA knockdown of ALDH1A1 (but not ALDH2) enhanced R848-induced production of TNFα (1.8-fold) in T-shNT cells but did not enhance TNFα overproduction in either FANCC-deficient (T-shFC) or FANCA-deficient (T-shFA [FANCA knockdown]) cells. Additionally, ALDH1A1 (but not ALDH2) mRNA was highly inducible in both THP-1 cells (by R848) and in Lin- Sca-1+ Kit+ murine marrow cells (by TNFα). While ALDH1A1 mRNA is expressed similarly in both T-shNT and T-shFC cells, our results indicate that in normal macrophages ALDH1A1 plays a role in the constraining TLR-induced TNF production but is significantly less functional in FANCC-deficient macrophages. In summary, (1) either pharmacological enhancement of ALDH function with Alda-1 or overexpression of ALDH1A1 is sufficient to restore the TLR-suppressive function of ALDH1A1 in FANCC-deficient macrophages, and (2) specific suppression of ALDH1A1, (but not ALDH2) induces an FA-like phenotype in control macrophages. We conclude that (1) optimal function of ALDH1A1 is FANCC- and FANCA-dependent in normal macrophages, (2) TLR-dependent overproduction of inflammatory cytokines by FA-deficient macrophages may result either from an increase in aldehyde load or the loss of a non-canonical signal-suppressive function of ALDH1A1, and (3) enhancement of ALDH activity using small molecule agonists such as Alda-1 may alleviate the FA macrophage/cytokine phenotype and thereby protect HSC from inflammation-induced exhaustion. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Gene Microarray Analyses of Daboia russelli russelli Daboiatoxin Treatment of THP-1 Human Macrophages Infected with Burkholderia pseudomallei.

Burkholderia pseudomallei is the causative agent of melioidosis and represents a potential bioterrorism threat. In this study, the transcriptomic responses of B. pseudomallei infection of a human macrophage cell model were investigated using whole-genome microarrays. Gene expression profiles were compared between infected THP-1 human monocytic leukemia cells with or without treatment with Daboia russelli russelli daboiatoxin (DRRDbTx) or ceftazidime (antibiotic control). Microarray analyses of infected and treated cells revealed differential upregulation of various inflammatory genes such as interleukin-1 (IL-1), IL-6, tumor necrosis factor-alpha (TNF-α), cyclooxygenase (COX-2), vascular endothelial growth factor (VEGF), chemokine C-X-C motif ligand 4 (CXCL4), transcription factor p65 (NF-kB); and several genes involved in immune and stress responses, cell cycle, and lipid metabolism. Moreover, following DRR-DbTx treatment of infected cells, there was enhanced expression of the tolllike receptor 2 (TLR-2) mediated signaling pathway involved in recognition and initiation of acute inflammatory responses. Importantly, we observed that highly inflammatory cytokine gene responses were similar in infected cells exposed to DRR-DbTx or ceftazidime after 24 h. Additionally, there were increased transcripts associated with cell death by caspase activation that can promote host tissue injury. In summary, the transcriptional responses during B. pseudomallei infection of macrophages highlight a broad range of innate immune mechanisms that are activated within 24 h post-infection. These data provide insights into the transcriptomic kinetics following DRR-DbTx treatment of human macrophages infected with B. pseudomallei.

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.

GW26-e0112 Curcumin Modulates Macrophage Polarization Through the Inhibition of the Toll-Like Receptor 4-Mitogen Activated Protein Kinase/Nuclear Factor-κB Pathways

Curcumin, the active ingredient in curcuma rhizomes, has a wide range of therapeutic effects. However, its atheroprotective activity in human acute monocytic leukemia THP-1 cells remains unclear. We investigated the activity and molecular mechanism of action of curcumin in polarized macrophages.

Autocrine motility factor receptor promotes the proliferation of human acute monocytic leukemia THP-1 cells.

The aberrant activation of autocrine motility factor receptor (AMFR) has been implicated in several types of human cancer. The present study aimed to elucidate the effect of AMFR on the regulation of proliferation in an acute monocytic leukemia cell line, THP-1. THP-1 cells were transfected with AMFR-targeted small interfering (si)RNA and a plasmid encoding a truncated AMFR, AMFR-C, (pcDNA3.1-AMFR-C). The mRNA and protein levels of AMFR and the downstream targets, rho-associated, coiled-coil containing protein kinase 2 (ROCK2), cyclin D1, and B-cell lymphoma (Bcl)-2, were measured using reverse transcription-quantitatibe polymerase chain reaction and immunoblot analyses. The effects on cell cycle and apoptosis were investigated using flow cytometry. The present study successfully established the knockdown of AMFR and expression of AMFR-C in the THP-1 cells. Downregulation of AMFR induced cell cycle arrest at the G0/G1 phase, and increased apoptosis of the THP-1 cells (all P<0.05). The AMFR siRNA increased the percentage of early apoptotic cells between 3.88±1.43 and 19.58±4.29% (P<0.05). The expression levels of ROCK2, cyclin D1 and Bcl-2 were reduced by the downregulation of AMFR and enhanced by overexpression of AMFR-C. In conclusion, AMFR appears to be crucial for the proliferation of the THP-1 acute monocytic leukemia cell line. Therefore, AMFR may represent a potential target for the treatment of acute monocytic leukemia.

The Effects of Phellinus linteus Polysaccharide Extracts on Cholesterol Efflux in Oxidized Low-Density Lipoprotein–Loaded THP-1 Macrophages

The removal of excess cellular cholesterol is critical for maintaining cellular cholesterol homeostasis. Phellinus linteus polysaccharide extracts (PLPEs) is an immunomudulatory agent with a molecular weight of 153 kd. Here,...

LZ-207, a Newly Synthesized Flavonoid, Induces Apoptosis and Suppresses Inflammation-Related Colon Cancer by Inhibiting the NF-κB Signaling Pathway.

Flavonoids and flavonoid derivatives, which have significant biological and pharmacological activities, including antitumor and anti-inflammatory activities, have been widely used in human healthcare. To design a more effective flavonoid antitumor agent, we altered the flavonoid backbone with substitutions of piperazine and methoxy groups to synthesize a novel flavonoid derivative, LZ-207. The anticancer effect of LZ-207 against HCT116 colon cancer cells and the underlying mechanism of this effect were explored in this study. Specifically, LZ-207 exhibited inhibitory effects on growth and viability in several human colon cancer cell lines and induced apoptosis in HCT116 cells both in vitro and in vivo. LZ-207 treatment also suppressed the nuclear translocation of NF-κB and the phosphorylation of IκB and IKKα/β in a dose-dependent manner in both HCT116 cells and human acute monocytic leukemia THP-1 cells. Moreover, LZ-207 also reduced the secretion of the pro-inflammatory cytokine interleukin-6 (IL-6) in LPS-induced THP-1 cells, and this effect was confirmed at the transcriptional level. Furthermore, LZ-207 significantly inhibited HCT116 cell proliferation that was elicited by LPS-induced THP-1 cells in a co-culture system. These findings elucidated some potential molecular mechanisms for preventing inflammation-driven colon cancer using the newly synthesized flavonoid LZ-207 and suggested the possibility of further developing novel therapeutic agents derived from flavonoids.

The Assembly of EDC4 and Dcp1a into Processing Bodies Is Critical for the Translational Regulation of IL-6

Macrophages play critical roles in the onset of various diseases and in maintaining homeostasis. There are several functional subsets, of which M1 and M2 macrophages are of particular interest because they are differentially involved in inflammation and its resolution. Here, we investigated the differences in regulatory mechanisms between M1- and M2-polarized macrophages by examining mRNA metabolic machineries such as stress granules (SGs) and processing bodies (P-bodies). Human monocytic leukemia THP-1 cells cultured under M1-polarizing conditions (M1-THPs) had less ability to assemble oxidative-stress-induced SGs than those cultured under M2-polarizing conditions (M2-THPs). In contrast, P-body assembly in response to oxidative stress or TLR4 stimulation was increased in M1-THPs as compared to M2-THPs. These results suggest that mRNA metabolism is controlled differently in M1-THPs and M2-THPs. Interestingly, knocking down EDC4 or Dcp1a, which are components of P-bodies, severely reduced the production of IL-6, but not TNF-α in M1-THPs without decreasing the amount of IL-6 mRNA. This is the first report to demonstrate that the assembly of EDC4 and Dcp1a into P-bodies is critical in the posttranscriptional regulation of IL-6. Thus, improving our understanding of the mechanisms governing mRNA metabolism by examining macrophage subtypes may lead to new therapeutic targets.

Screening natural product extracts against kinetoplastid parasites

The kinetoplastids parasites are a group of flagellated protozoan parasites characterized by the subcellular structure, kinetoplast. Trypanosoma spp and Leishmania spp are the most notable kinetoplastids with major impact on human and animal health. Human African Trypanosomiasis (HAT) caused by Trypansoma brucei and visceral leishmaniasis caused by Leishmania donovani affect mostly neglected segments of the populations. The toxicity and resistance for currently available drugs and depleted drug discovery pipeline, necessitate discovery of new drugs. Natural products are vast source of diverse drug molecules with novel chemotypes. A 384 well plate screening paradigm has been developed that covers the screening of natural products extracts against L. donovani promastigotes, L. donovani axenic amastigote, differentiated THP1 (Human Acute Monocytic Leukemia) cells infected with L. donovani amastigotes and also cytotoxicity against uninfected THP1 cells. In the initial phase 176 plant extracts, prepared from the North American plants, have been screened. Lead extracts Cupressus lawsoniana, Acer pensylvanicum, Verbesina virginica, were identified as new antitrypanosomal leads with significant selectivity. Further follow up evaluation of lead extracts is likely to identify drug leads with novel chemotypes.

Ramalin inhibits VCAM-1 expression and adhesion of monocyte to vascular smooth muscle cells through MAPK and PADI4-dependent NF-kB and AP-1 pathways.

Cell adhesion molecules play a critical role in inflammatory processes and atherosclerosis. In this study, we investigated the effect of ramalin, a chemical compound from the Antarctic lichen Ramalina terebrata, on vascular cell adhesion molecule-1 (VCAM-1) expression induced by TNF-α in vascular smooth muscular cells (VSMCs). Pretreatment of VSMCs with ramalin (0.1-10μg/mL) concentration-dependently inhibited TNF-α-induced VCAM-1 expression. Additionally, ramalin inhibited THP-1 (human acute monocytic leukemia cell line) cell adhesion to TNF-α-stimulated VSMCs. Ramalin suppressed TNF-α-induced production of reactive oxygen species (ROS), PADI4 expression, and phosphorylation of p38, ERK, and JNK. Moreover, ramalin inhibited TNF-α-induced translocation of NF-κB and AP-1. Inhibition of PADI4 expression by small interfering RNA or the PADI4-specific inhibitor markedly attenuated TNF-α-induced activation of NF-κB and AP-1 and VCAM-1 expression in VSMCs. Our study provides insight into the mechanisms underlying ramalin activity and suggests that ramalin may be a potential therapeutic agent to modulate inflammation within atherosclerosis.

Circulating microparticles carry oxidation-specific epitopes and are recognized by natural IgM antibodies

Oxidation-specific epitopes (OSEs) present on apoptotic cells and oxidized low density lipoprotein (OxLDL) represent danger-associated molecular patterns that are recognized by different arcs of innate immunity, including natural IgM antibodies. Here, we investigated whether circulating microparticles (MPs), which are small membrane vesicles released by apoptotic or activated cells, are physiological carriers of OSEs. OSEs on circulating MPs isolated from healthy donors and patients with ST-segment elevation myocardial infarction (STE-MI) were characterized by flow cytometry using a panel of OSE-specific monoclonal antibodies. We found that a subset of MPs carry OSEs on their surface, predominantly malondialdehyde (MDA) epitopes. Consistent with this, a majority of IgM antibodies bound on the surface of circulating MPs were found to have specificity for MDA-modified LDL. Moreover, we show that MPs can stimulate THP-1 (human acute monocytic leukemia cell line) and human primary monocytes to produce interleukin 8, which can be inhibited by a monoclonal IgM with specificity for MDA epitopes. Finally, we show that MDA+ MPs are elevated at the culprit lesion site of patients with STE-MI. Our results identify a subset of OSE+ MPs that are bound by OxLDL-specific IgM. These findings demonstrate a novel mechanism by which anti-OxLDL IgM antibodies could mediate protective functions in CVD.

Cyr61 promotes CD204 expression and the migration of macrophages via MEK/ERK pathway in esophageal squamous cell carcinoma.

Tumor-associated macrophages (TAMs) are known to be involved in the progression of various human malignancies. We previously demonstrated that CD204 was a useful marker for TAMs contributing to the angiogenesis, progression, and prognosis of human esophageal squamous cell carcinoma (ESCC). We also showed that conditioned media of ESCC cell lines induced CD204 expression in THP-1 human monocytic leukemia cells. Here, we performed a cDNA microarray analysis between THP-1 cells stimulated with TPA (macrophage [MΦ]-like THP-1 cells) treated with and without conditioned medium of ESCC cell line to clarify the molecular characteristics of TAMs in ESCC. From the microarray data, we discovered that Cyr61 was induced in CD204-positive-differentiated THP-1 cells (TAM-like THP-1 cells). In the ESCC microenvironment, not only cancer cells but also TAMs expressed Cyr61. Interestingly, the expression levels of Cyr61 showed a significant positive correlation with the number of CD204-positive macrophages in ESCCs by immunohistochemistry. Recombinant human Cyr61 (rhCyr61) promoted cell migration and induced the expression of CD204 along with the activation of the MEK/ERK pathway in MΦ-like THP-1 cells. Pretreatment with a MEK1/2 inhibitor significantly inhibited not only the Cyr61-mediated migration but also the CD204 expression in the MΦ-like THP-1 cells. These results suggest that Cyr61 may contribute to the expression of CD204 and the promotion of cell migration via the MEK/ERK pathway in TAMs in the ESCC microenvironment.

Flavonoid 4'-O-Methylkuwanon E from Morus alba Induces the Differentiation of THP-1 Human Leukemia Cells.

Aims. In this work we studied cytodifferentiation effects of newly characterized prenyl flavonoid 4′-O-methylkuwanon E (4ME) isolated from white mulberry (Morus alba L.). Main Methods. Cell growth and viability were measured by dye exclusion assay; cell cycle and surface antigen CD11b were monitored by flow cytometry. For the cytodifferentiation of cells the NBT reduction assay was employed. Regulatory proteins were assessed by western blotting. Key Findings. 4ME induced dose-dependent growth inhibition of THP-1 cells, which was not accompanied by toxic effect. Inhibition of cells proliferation caused by 4ME was associated with the accumulation in G1 phase and with downregulation of hyperphosphorylated pRb. Treatment with 4ME led to significant induction of NBT-reducing activity of PMA stimulated THP-1 cells and upregulation expression of differentiation-associated surface antigen CD11b. Our results suggest that monocytic differentiation induced by 4ME is connected with up-regulation of p38 kinase activity. Significance. Our study provides the first evidence that 4ME induces the differentiation of THP-1 human monocytic leukemia cells and thus is a potential cytodifferentiating anticancer agent.

Curcumin Modulates Macrophage Polarization Through the Inhibition of the Toll-Like Receptor 4 Expression and its Signaling Pathways

Background: Curcumin, the active ingredient in curcuma rhizomes, has a wide range of therapeutic effects. However, its atheroprotective activity in human acute monocytic leukemia THP-1 cells remains unclear. We investigated the activity and molecular mechanism of action of curcumin in polarized macrophages. Methods: Phorbol myristate acetate (PMA)-treated THP-1 cells were differentiated to macrophages, which were further polarized to M1 cells by lipopolysaccharide (LPS; 1 µg/ml) and interferon (IFN)-γ (20 ng/ml) and treated with varying curcumin concentrations. [³H]thymidine (³H-TdR) incorporation assays were utilized to measure curcumin-induced growth inhibition. The expression of tumor necrosis factor-a (TNF-a), interleukin (IL-6), and IL-12B (p40) were measured by quantitative real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). Macrophage polarization and its mechanism were evaluated by flow cytometry and western blot. Additionally, toll-like receptor 4 (TLR4) small interfering RNA and mitogen-activated protein kinase (MAPK) inhibitors were used to further confirm the molecular mechanism of curcumin on macrophage polarization. Results: Curcumin dose-dependently inhibited M1 macrophage polarization and the production of TNF-a, IL-6, and IL-12B (p40). It also decreased TLR4 expression, which regulates M1 macrophage polarization. Furthermore, curcumin significantly inhibited the phosphorylation of ERK, JNK, p38, and nuclear factor (NF)-γB. In contrast, SiTLR4 in combination with p-JNK, p-ERK, and p-p38 inhibition reduced the effect of curcumin on polarization. Conclusions: Curcumin can modulate macrophage polarization through TLR4-mediated signaling pathway inhibition, indicating that its effect on macrophage polarization is related to its anti-inflammatory and atheroprotective effects. Our data suggest that curcumin could be used as a therapeutic agent in atherosclerosis.

ALDH1A1 Dysfunction in FA-Deficient Macrophages Contributes to Aberrant Activation of the Innate Immune Response

Recent work has clarified a role for aldehyde dehydrogenases in protecting Fanconi anemia (FA) hematopoietic stem cells (HSC) and indicates that the increase in endogenous aldehydes that attends the genetic loss of ALDH function is sufficient to induce bone marrow failure in this disease. Studies in many laboratories have also documented that: (1) exposure of FA HSC to inflammatory cytokines suppresses stem cell self-replication and, in vivo, exhausts the stem cell pool and (2) FA macrophages exposed to specific toll-like receptor (TLR) ligands overproduce the very inflammatory cytokines that suppress the HSC pool. Given that aldehydes form adducts with and, in some cases, activate signaling proteins involved in cytokine production, we tested the hypothesis that overproduction of inflammatory cytokines by FA macrophages results from a loss of FA protein-dependent ALDH function. We treated THP-1 human monocytic leukemia cells expressing shRNA targeting FANCC (T-shFC) or a non-targeted shRNA (T-shNT) with the aldehyde 4-hydroxynonenal (4-HNE) before exposing them to the TLR-7/8 agonist R848. 4-HNE alone did not induce TNF production in either cell line but did enhance TLR-induced TNF overproduction by T-shFC cells (but not T-shNT cells), suggesting that FANCC-deficient macrophages lack factors (e.g., ALDH) that neutralize a signal-enhancing effect of this aldehyde. To directly test the effects of ALDH loss, we pretreated cells with the general ALDH inhibitor diethylaminobenzaldehyde and found that this agent enhanced R848-induced normal production of TNF by approximately 1.6-fold in control cells but did not enhance TNF production in T-shFC cells that were already overproducing TNF. Having determined that ALDH1A1 (but not ALDH2) was highly inducible in both THP-1 cells (by R848) and Lin-Sca-1+Kit+ (by TNF) murine marrow cells, we used siRNA to suppress expression of ALDH2 or ALDH1A1. Knockdown of ALDH1A1 (but not ALDH2) enhanced R848-induced normal production of TNF by approximately 1.8-fold in T-shNT but not TNF overproduction in T-shFC (fig 1) or T-shFA (FANCA knockdown) cells. Our results are consistent with the notion that ALDH1A1 is non-functional in FANCC-deficient macrophages and we confirmed that suspicion in gain-of-function analyses. Specifically, treatment of T-shNT and T-shFC cells with Alda-1 (a small molecule ALDH agonist known to enhance the activity of both ALDH1A1 and ALDH2) suppressed TLR-induced TNF production (fig 2), even in the presence of 4-HNE, by both T-shNT and T-shFC cells. In summary, (1) increasing the aldehyde load in normal macrophages has little influences on the inflammatory response induced by TLR activation but in FANCC-deficient cells aldehydes exacerbate the inflammatory response, (2) suppression of ALDH function with DEAB and specific suppression of ALDH1A1, (but not ALDH2) induces an FA-like phenotype in control macrophages, and (3) pharmacological enhancement of ALDH activity suppresses induced cytokine overproduction by FANCC-deficient macrophages. We conclude that: (1) optimal function of ALDH1A1 is FANCC-dependent in normal macrophages, (2) that the TLR-dependent overproduction of inflammatory cytokines by FANCC-deficient macrophages may result either from an increase in the aldehyde load or the loss of a non-canonical signal-suppressive function of ALDH1A1, and (3) enhancement of ALDH activity using small molecule agonists such as Alda-1 may alleviate the FA macrophage phenotype and may thereby protect HSC from inflammation-induced exhaustion. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

In vitro study of the anti-inflammatory activity of some medicinal and edible plants growing in Russia

The effects of the ethanolic extracts of 105 plants used in Russian traditional medicine, 26 vegetables and fruits and 2 mushrooms on the release of tumor necrosis factor α (TNF-α) and prostaglandin E2 (PGE2) in lipopolysaccharide (LPS)-stimulated differentiated human acute monocytic leukemia THP-1 cells were studied using TNF-α and PGE2 assays, respectively. We found that 16 plant extracts inhibited TNF-α production and 15 extracts decreased PGE2 release in the cells in a concentration-dependent manner. Guilder rose, tansy, shrubby cinquefoil, wintergreen, and prince’s pine, the last two of which belong to the Pyrolaceae family, notably inhibited the levels of both of the inflammatory mediators.