PMA-treated THP-1 Cells Research Articles

Characterization of a Myeloid Differentiation Factor 2-Derived Peptide that Facilitates THP-1 Macrophage-Mediated Phagocytosis of Gram-Negative Bacteria.

Myeloid differentiation factor 2 (MD2), the TLR4 coreceptor, has been shown to possess opsonic activity and has been implicated in phagocytosis and intracellular killing of Gram-negative bacteria. However, any MD2 protein segment involved in phagocytosis of Gram-negative bacteria is not yet known. A short synthetic MD2 segment, MD54 (amino acid regions 54 to 69), was shown to interact with a Gram-negative bacterial outer membrane component, LPS, earlier. Furthermore, the MD54 peptide induced aggregation of LPS and facilitated its internalization in THP-1 cells. Currently, it has been investigated if MD2-derived MD54 possesses any opsonic property and role in phagocytosis of Gram-negative bacteria. Remarkably, we observed that MD54 facilitated agglutination of Gram-negative bacteria, Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC BAA-427), but not of Gram-positive bacteria, Bacillus subtilis (ATCC 6633) and Staphylococcus aureus (ATCC 25923). The MD54-opsonized Gram-negative bacteria internalized within PMA-treated THP-1 cells and were killed over a longer incubation period. However, both internalization and intracellular killing of the MD54-opsonized Gram-negative bacteria within THP-1 phagocytes were appreciably inhibited in the presence of a phagocytosis inhibitor, cytochalasin D. Furthermore, MD54 facilitated the clearance of Gram-negative bacteria E. coli (ATCC 25922) and P. aeruginosa (ATCC BAA-427) from the infected BALB/c mice whereas an MD54 analog, MMD54, was inactive. Overall, for the first time, the results revealed that a short MD2-derived peptide can specifically agglutinate Gram-negative bacteria, act as an opsonin for these bacteria, and facilitate their phagocytosis by THP-1 phagocytes. The results suggest that the MD54 segment could have a crucial role in MD2-mediated host-pathogen interaction involving the Gram-negative bacteria.

Gastric cancer-derived LBP promotes liver metastasis by driving intrahepatic fibrotic pre-metastatic niche formation

BackgroundLiver metastasis (LM) is one of the most common distant metastases of gastric cancer (GC). However, the mechanisms underlying the LM of GC (GC-LM) remain poorly understood. This study aimed to identify the tumour-secreted protein associated with GC-LM and to investigate the mechanisms by which this secreted protein remodels the liver microenvironment to promote GC-LM.MethodsData-independent acquisition mass spectrometry (DIA-MS), mRNA expression microarray, quantitative real-time PCR, enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry (IHC) were performed to identify and validate the GC-secreted proteins associated with GC-LM. A modified intrasplenic injection mouse model of LM was used to evaluate the progression and tumour burden of LM in vivo. Flow cytometry, immunofluorescence (IF), western blots (WB) and IHC were performed to validate the pre-metastatic niche (PMN) formation in the pre-modelling mouse models. mRNA sequencing of PMA-treated THP-1 cells with or without lipopolysaccharide binding protein (LBP) treatment was used to identify the functional target genes of LBP in macrophages. Co-immunoprecipitation (Co-IP), WB, ELISA, IF and Transwell assays were performed to explore the underlying mechanism of LBP in inducing intrahepatic PMN formation.ResultsLBP was identified as a critical secreted protein associated with GC-LM and correlated with a worse prognosis in patients with GC. LBP activated the TLR4/NF-κB pathway to promote TGF-β1 secretion in intrahepatic macrophages, which, in turn, activated hepatic satellite cells (HSCs) to direct intrahepatic fibrotic PMN formation. Additionally, TGF-β1 enhanced the migration and invasion of incoming metastatic GC cells in the liver. Consequently, selective targeting of the TGF-β/Smad signaling pathway with galunisertib demonstrated its efficacy in effectively preventing GC-LM in vivo.ConclusionsThe results of this study provide compelling evidence that serological LBP can serve as a valuable diagnostic biomarker for the early detection of GC-LM. Mechanistically, GC-derived LBP mediates the crosstalk between primary GC cells and the intrahepatic microenvironment by promoting TGF-β1 secretion in intrahepatic macrophages, which induces intrahepatic fibrotic PMN formation to promote GC-LM. Importantly, selectively targeting the TGF-β/Smad signaling pathway with galunisertib represents a promising preventive and therapeutic strategy for GC-LM.

Role of ICAM1 in tumor immunity and prognosis of triple-negative breast cancer.

Treating triple-negative breast cancer (TNBC) is a difficult landscape owing to its short survival times and high risk of metastasis and recurrence among patients. Although involved in tumor invasion and metastasis, the mechanism of action of intercellular adhesion molecule 1 (ICAM1), a trans-membrane glycoprotein, in TNBC is ambiguous. We examined ICAM1's role in TNBC, focusing on its expression, cell survival, mutation, and tumor immunity. Then, a risk score model was created utilizing co-expressed genes associated with ICAM1. According to their respective risk scores, we divided patients into high- and low-risk groups. Immune function, drug susceptibility differences, and somatic variants were analyzed in the high-and low-risk groups. And we used the CMap database to predict potential medications. Then, TNBC cells with low expression of ICAM-1 were co-cultured with PMA-treated THP-1 cells and CD8 T cells. In addition, We detected the expression of PD-1 and CTLA4 of low ICAM-1 expressing TNBC cells when they were cocultured with CD8 T cells. ICAM1 was found to be involved in leukocyte cell adhesion, motility, and immune activation. Patients with low-ICAM1 group had shorter disease-free survival (DFS) than those with high-ICAM1 group. The group with elevated levels of ICAM1 exhibited significantly increased levels of T-cell regulation, quiescence in natural killer (NK) cells, and M1 macrophage. ICAM1 expression was correlated with immune checkpoint drugs. The prognostic ability of the risk score model was found to be superior to that of individual genes. Patients categorized as high-risk exhibited elevated clinical stages, showed higher M1 macrophage numbers, and were able to benefit better from immunotherapy. Individuals belonging to the high-risk group exhibit significantly elevated mutation rates in TP53, TTN, and SYNE1 genes, along with increased TMB and PD-L1 levels and decreased TIDE scores. These findings suggest that immunotherapy may be advantageous for the high-risk group. Furthermore, low expression of ICAM1 was found to promote polarization to M2 macrophages along with T-cell exhaustion. In conclusion, Low ICAM1 expression may be related to immune escape, leading to poor treatment response and a worse prognosis.

Laeverin/aminopeptidase Q induces indoleamine 2,3-dioxygenase-1 in human monocytes

Human extravillous trophoblast (EVT) invades the maternal endometrium and reconstructs uterine spiral arteries cooperatively with maternal immune cells. Although EVT has allogeneic paternal antigens, the maternal immune system does not reject it. Here, we found that laeverin (LVRN), an EVT-specific cell surface peptidase, interacts with monocytes to produce indoleamine 2,3-dioxygenase-1 (IDO1). LVRN-transfected Swan71 cells, a cytotrophoblast-derived cell line, and increased IDO1 expression in PBMC under cell-to-cell interacting conditions. Soluble recombinant LVRN (r-LVRN) interacted with CD14-positive monocytes and induced their IDO1 expression without the intervention of other immune cell populations. LVRN-induced IDO1 production was promoted in PMA-activated monocyte-like THP-1 cells. Furthermore, r-LVRN decreased the tryptophan level and increased the kynurenine/tryptophan ratio in the culture media of the PMA-treated THP-1 cells. These findings suggest that LVRN is one of the key molecules that mediate the interaction between EVT and monocytes/macrophages and creates an immunosuppressive environment at the maternal-fetal interface in the uterus.

Gastric cancer-derived exosomal miR-519a-3p promotes liver metastasis by inducing intrahepatic M2-like macrophage-mediated angiogenesis

BackgroundLiver metastasis (LM) is a major obstacle to the prognosis of gastric cancer (GC) patients, but the molecular mechanism underlying gastric cancer liver metastasis (GC-LM) remains unknown. Exosomes have been identified as an important mediator of communication between tumor cells and the microenvironment. Therefore, we sought to investigate the effects of primary GC cells on the liver microenvironment and the role of exosomal microRNAs (exo-miRNA) in GC-LM.MethodsSequential differential centrifugation, transmission electron microscopy and NanoSight analysis were used to extract and characterize exosomes. MicroRNA sequencing in GC-derived exosomes and mRNA sequencing in PMA-treated THP-1 cells were used to identify differentially expressed miRNAs in exosomes and the functional targets of exosomal miR-519a-3p (exo-miR-519a-3p) in macrophages, respectively. Tracing and internalization of exosomes and transfer of exo-miR-519a-3p were observed by immunofluorescence. Tubule formation assays, aortic ring assays, and exosome-educated GC-LM model were used to investigate the roles of GC-derived exosomes and exo-miR-519a-3p in angiogenesis and GC-LM. Luciferase reporter assay, qRT-PCR, Western blot, ELISA, flow cytometry and immunofluorescence were used to investigate the regulatory mechanism of exo-miR-519a-3p at GC-LM.ResultsThe expression level of miR-519a-3p in serum exosomes was significantly higher in GC-LM patients than in patients without LM, and high expression of exo-miR-519a-3p indicates a worse prognosis. GC-derived exosomes are mainly accumulated in the liver and internalized by intrahepatic macrophages. Mechanistically, exo-miR-519a-3p activates the MAPK/ERK pathway by targeting DUSP2, thereby causing M2-like polarization of macrophages. M2-like polarized macrophages accelerate GC-LM by inducing angiogenesis and promoting intrahepatic premetastatic niche formation.ConclusionsOur results indicate that exo-miR-519a-3p plays a critical role in mediating crosstalk between primary GC cells and intrahepatic macrophages and is a potential therapeutic target for GC-LM.

PAQR3 depletion accelerates diabetic wound healing by promoting angiogenesis through inhibiting STUB1-mediated PPARγ degradation

AbstractThe pathogenesis of diabetic wounds is closely associated with the dysregulation of macrophage polarization. However, the underlying mechanism remains poorly understood. In this study, we aimed to investigate the potential effects of PAQR3 (progestin and adipoQ receptor 3) silencing in accelerating diabetic wound healing. We showed that PAQR3 silencing promoted skin wound healing and angiogenesis in diabetic mice, which was accompanied by enhanced M2 macrophage polarization and elevated expression of PPARγ (peroxisome proliferator-activated receptor γ). PAQR3 silencing also promoted M2 polarization and increased PPARγ protein level in PMA-treated THP-1 cells. Moreover, knockdown of PAQR3 in macrophages enhanced the migration of HaCaT cells and tube formation of HUVECs. The ubiquitination of PPARγ protein in macrophages was repressed by PAQR3 silencing. STUB1 (STIP1 homology and U-box-containing protein 1) binds with the PPARγ protein to mediate PPARγ ubiquitination and degradation in macrophages, which was impaired by PAQR3 silencing. The PPARγ inhibitor, GW9662, or STUB1 overexpression abrogated the enhanced M2 macrophage polarization induced by PAQR3 silencing. Therefore, these findings demonstrates that PAQR3 silencing accelerates diabetic wound healing by promoting M2 macrophage polarization and angiogenesis, which is mediated by the inhibition of STUB1-mediated PPARγ protein ubiquitination and degradation.

Interferon Gamma-Induced Interferon Regulatory Factor 1 Activates Transcription of HHLA2 and Induces Immune Escape of Hepatocellular Carcinoma Cells.

Immunosuppression developed by cancer cells remains a leading cause of treating failure of immunotherapies. This study aimed to explore the function of human endogenous retrovirus-H long terminal repeat-associating 2 (HHLA2), an immune checkpoint molecule from the B7 family, in the immune escape in hepatocellular carcinoma (HCC). Mouse models with primary HCC or with xenograft tumors were established. The portion of tumor-associated macrophages (TAMs) and the level of PD-L1 in the tumor tissues were examined. THP-1 cells were treated with PMA to obtain a macrophage-like phenotype. The PMA-treated THP-1 cells were co-cultured with the HCC cells in Transwell chambers to examine the function of HHLA2 in chemotactic migration and polarization of macrophages. HHLA2 expression was correlated with infiltration of immune cells, especially macrophages, and was linked to poor prognosis of patients with HCC. HHLA2 knockdown reduced incidence rate of primary HCC in mice. It also reduced tumor metastasis, the portion of M2 macrophages, and the expression of PD-L1 in primary and xenograft tumors. In vitro, HHLA2 upregulation increased expression of PD-L1 in HCC cells indirectly by inducing M2 polarization and chemotactic migration of macrophages. Interferon gamma (IFNG) enhanced expression of interferon regulatory factor 1 (IFR1) in HCC cells, and IFR1 bound to the promoter region of HHLA2 to activate HHLA2 expression. This study suggested that the IFNG/IFR1/HHLA2 axis in HCC induces M2 polarization and chemotactic migration of macrophages, which leads to immune escape and development of HCC.

A fluorometric erythrophagocytosis assay using differentiated monocytic THP-1 cells to assess the clinical significance of antibodies to red blood cells.

The significance of antibodies to red blood cells (RBCs) is variable and cannot be predicted solely by serological testing. A flow cytometry-based erythrophagocytosis assay was established using phorbol 12-myristate 13-acetate (PMA)-treated THP-1 cells and RBCs labelled with PKH26 to assess allo- and autoantibodies to RBCs. THP-1 cells were differentiated into macrophage-like cells by treatment with PMA. RBC samples coated with alloantibodies or autoantibodies were obtained from 16 patients with autoimmune haemolytic anaemia of warm type (wAIHA) as well as from five pregnant women with warm autoantibodies. RBCs from healthy blood donors were used as controls. RBCs were labelled with the red lipophilic fluorescent dye PKH26 and incubated with PMA-treated THP-1 cells. After removal of nonadherent RBCs by washing and haemolysis of adherent RBCs, erythrophagocytosis was quantified by flow cytometry. We observed significant phagocytosis of RBCs coated with clinically relevant alloantibodies (i.e. anti-D and anti-K) or autoantibodies from patients with active wAIHA, but not of those coated with alloantibodies (anti-Ch) or autoantibodies from patients and pregnant women without haemolysis. The flow cytometry-based erythrophagocytosis test described here is quantitative, highly reliable, and may be helpful for the assessment of the clinical significance of antibodies to RBCs.

Inhibition of inflammatory-molecule synthesis in THP-1 cells stimulated with phorbol 12-myristate 13-acetate by brefelamide derivatives

Plasma osteopontin (OPN) levels are elevated in tuberculosis patients and may involve granuloma formation. New inhibitors using brefelamide, an aromatic amide isolated from Dictyostelium cellular slime molds that may inhibit OPN transcription in A549 cells at 1 μM concentration, were synthesized as compounds C, D, and E. Their inhibitory activity against OPN synthesis in phorbol 12-myristate 13-acetate (PMA)-stimulated THP-1 cells was confirmed using enzyme-linked immunosorbent assay (ELISA), a multicolor immune-fluorescent microscope, and western blot. In the ELISA performed using full-length OPN, each compound showed significant inhibition in culture supernatants with half maximal inhibitory concentration (IC50) values of 1.6, 1.8, and 2.2 μM for C, D, and E, respectively. In another ELISA to detect the immune-related form of OPN, IC50 values were 0.6, 1.2, and 2.5 μM for compounds C, D, and E, respectively. The decreases in OPN expression and synthesis were confirmed using immunofluorescence and western blot studies using compound-treated cells or cell lysates. Luminex assay of the supernatants of PMA-treated THP-1 cells showed significant reduction in the synthesis of interleukin (IL)-1β, galectin-9, and tumor necrosis factor (TNF)-α. Elucidation of the detailed mechanisms of the biological activities of these compounds would be necessary; however, they may be used in clinical trials for infectious diseases, inflammatory disorders, and cancer.

Chemotherapy agents induce tartrate-resistant acid phosphatase 5a contributing to the symptom distress in lung cancer patients

Tartrate-resistant acid phosphatase 5a (TRACP5a) is mainly secreted by activated macrophages in chronic inflammation. Serum TRACP5a is associated with symptom distress in lung cancer patients during chemotherapy. Therefore, this study aimed to investigate whether chemotherapy drugs modulate TRACP5a as an inducible marker for symptom distress in lung cancer patients during chemotherapy. In clinical analysis, lung cancer participants completely received the six-cycle chemotherapy process (n = 42). Clinical determinations for TRACP5a, C-reactive protein (CRP), interleukin-6 (IL-6), white blood cells, monocytes, and hemoglobin were analyzed at six time points: BL, C1d8, C2d1, C4d1, C4d8, and Ed28. Meanwhile, five questionnaires for fatigue, sleep disturbance, pain, depression, and confusion were finished before drug treatment. For monocyte-to-macrophage differentiation, THP-1 cells were treated with phorbol 12-myristate 13-acetate (PMA). TRACP5a secretion in THP-1 cells was determined at the following days up to 6 days after 1-day incubation of chemotherapy drugs by dot blotting. Clinical analysis revealed that TRACP5a significantly increased at C1d8 and C4d8, but dropped at C2d1 and Ed28. CRP and IL-6 displayed a broad-range variation, resulting in no significant difference among the assessment time points. In contrast, monocytes decreased at C1d8 and C4d8, but rose again at C2d1 and Ed28. In symptom distress, the changes only in fatigue and sleep disturbance were positively associated with the trend in TRACP5a. In PMA-treated THP-1 cells, TRACP5a significantly increased after stimulation with gemcitabine and paclitaxel. Taken together, induction of TRACP5a by chemotherapy drugs might be generated from monocyte-differentiated macrophages, further causing clinical symptom distress in lung cancer patients.

Sp1 mediates phorbol ester (PMA)-induced expression of membrane-bound guanylyl cyclase GC-A in human monocytic THP-1 cells.

Cyclic guanosine monophosphate (cGMP) is synthesized by two types of enzymes: particulate (membrane-bound) guanylyl cyclases (pGCs) and soluble (cytosolic) guanylyl cyclases (sGCs). sGCs are primarily activated by binding of nitric oxide to their prosthetic heme group while pGCs are activated by binding of peptide ligands to their extracellular domains. One of them, pGC type A (GC-A) is activated by atrial and brain natriuretic peptides (ANP and BNP, respectively). Human monocytes isolated from peripheral blood mononuclear cells have been found to display sGC expression without concomitant expression of GC-A. However, GC-A activity appears in monocytes under certain conditions but a molecular mechanism of GC-A expression is still poorly understood. In this report we show that phorbol ester (PMA) induces transcription of a gene encoding GC-A in human monocytic THP-1 cells. Moreover, we find that PMA-treated THP-1 cells raise cGMP content following treatment with ANP. Studies using pharmacological inhibitors of protein kinases suggest involvement of protein kinase C (PKC), mitogen extracellular kinases (MEK1/2), and extracellular signal-regulated kinases (ERK1/2) in PMA-induced expression of the GC-A encoding gene in THP-1 cells. Finally, we show that PMA stimulates binding of Sp1 transcription factor to GC-rich DNA sequences and mithramycin A (a selective Sp1 inhibitor) inhibits expression of the GC-A mRNA in PMA-treated THP-1 cells. Taken together, our findings suggest that the PMA-stimulated PKC and MEK/ERK signaling pathways induce Sp1-mediated transcription of the GC-A encoding gene in human monocytic THP-1 cells.

MicroRNA-24 aggravates atherosclerosis by inhibiting selective lipid uptake from HDL cholesterol via the post-transcriptional repression of scavenger receptor class B type I

Background and aimsLiver scavenger receptor class B type I (SR-BI) exerts atheroprotective effects through selective lipid uptake (SLU) from high-density lipoprotein cholesterol (HDL-C). Low hepatic SR-BI expression leads to high HDL-C levels in the circulation and an increased risk of atherosclerosis. Furthermore, macrophage SR-BI mediates bidirectional cholesterol flux and may protect against atherogenesis. Previous studies have revealed that miR-24 is closely related to cardiovascular disease (CVD) progression. We aimed to investigate the molecular mechanisms by which miR-24 participates in SR-BI-mediated selective HDL cholesteryl ester (HDL-CE) uptake and further atherogenesis in apoE−/− mice. MethodsBioinformatic predictions and luciferase reporter assays were utilized to detect the association between miR-24 and the SR-BI 3′ untranslated region (3′ UTR), and RT-PCR and western blotting were used to evaluate SR-BI mRNA and protein expression, respectively. The effects of miR-24 on Dil-HDL uptake were determined by flow cytometry assay. Double-radiolabeled HDL (125I-TC-/[3H] CEt-HDL) was utilized to measure the effects of miR-24 on HDL and CE binding and SLU in HepG2 and PMA-treated THP-1 cells. In addition, total cholesterol (TC) levels in HepG2 cells were analyzed using enzymatic methods, and macrophage lipid content was evaluated by high-performance liquid chromatography (HPLC) assay. Small interfering RNA (siRNA) and pcDNA3.1(−)-hSR-BI plasmid transfection procedures were utilized to confirm the role of SR-BI in the effects of miR-24 on Dil-HDL uptake, SLU and cholesterol levels in both cell types. Hepatic SR-BI level in apoE−/− mice was measured by western blotting. Liver TC, FC and CE levels and plasma triglycerides (TG), TC and HDL-C levels were evaluated enzymatically using commercial test kits. Atherosclerotic lesion sizes were measured using Oil Red O and hematoxylin-eosin staining. ResultsmiR-24 directly repressed SR-BI expression by targeting its 3′UTR. In addition, miR-24 decreased Dil-HDL uptake and SLU in HepG2 and THP-1 macrophages. In the presence of HDL, miR-24 decreased TC levels in HepG2 cells and TC, free cholesterol (FC) and CE levels in macrophages. Overexpression and down-regulation assays showed that SR-BI mediated the effects of miR-24 on Dil-HDL uptake, SLU and cholesterol levels. Lastly, miR-24 administration decreased hepatic SR-BI expression and promoted atheromatous plaque formation in apoE−/− mice, findings in line with those of our in vitro studies. ConclusionsThese findings indicate that miR-24 accelerates atherogenesis by repressing SR-BI-mediated SLU from HDL-C.

Upregulation of PD-L1 by SPP1 mediates macrophage polarization and facilitates immune escape in lung adenocarcinoma

Tumor-associated macrophages (TAMs) polarization represents a key regulatory process of tumor progression. However, the underlying mechanisms are unclear. This study aimed to investigate the relationship between secreted phosphoprotein 1 (SPP1) and TAMs in lung adenocarcinoma cells. THP-1 monocytes were differentiated into macrophages using PMA. PMA-treated THP-1 cells were co-cultured with human A549 cells culture supernatant. SPP1 expression in TAMs isolated from lung adenocarcinoma tissues and PMA-treated THP-1 cells were measured. Macrophage polarization was identified by flow cytometric analysis. Cell migration and apoptosis were assessed by Transwell migration assays and flow cytometric analysis, respectively. SPP1 is highly expressed in tumor tissues and TAMs isolated from patients with an advanced TNM stage, and also in PMA-treated THP-1 cells. Co-culture with A549 cells strongly induced SPP-1 expression as well as M2 polarization of THP-1 cells, but it had little effect on short hairpin SPP1 (shSPP1)-transfected THP-1 cells. Interestingly, programmed death ligand 1 (PD-L1), a critical regulator of M2 polarization, was downregulated in SPP1 knockdown THP-1 cells. Inhibition of PD-L1 induced a greater decline of the M2 markers IL-10 and Arg-1 but an increase in the M1 markers IL-12 and TNF-α. In addition, SPP1 knockdown in THP-1 cells can mitigate migration but promote apoptosis of A549 cells, and PD-L1 inhibition can further enhance this effect. THP-1 cells co-cultured with A549 cells attenuated CD4+ T-cell activation, whereas SPP1 inhibition restored T-cell activation. These results highlight the importance of SPP1 in mediating macrophage polarization and lung cancer evasion, suggesting a potential therapeutic target for lung cancer.

PI3K/Akt regulates survival during differentiation of human macrophages by maintaining NF-κB-dependent expression of antiapoptotic Bcl-xL

Resistance to apoptosis is an important characteristic that human macrophages acquire during differentiation from monocytes. However, the intracellular mechanisms that mediate the development of resistance are not well understood. We have used M-CSF-stimulated primary human monocytes and PMA-treated THP1 cells to study apoptosis resistance during differentiation of human macrophages. Our results indicate that PI3K/Akt distinctively regulates survival of macrophages during and after differentiation. More specifically, a signaling pathway consisting of PI3K/Akt-NF-κB-Bcl-xL regulates cell survival during the differentiation process. PI3K/Akt-mediated activation of NF-κB plays a key role in survival of differentiating macrophages by specifically sustaining antiapoptotic Bcl-xL expression. With the use of pharmacological inhibitors and siRNA for Akt and Bcl-xL, we show that in the absence of Akt-dependent Bcl-xL expression during differentiation, cells undergo caspase-mediated apoptosis. In contrast, in differentiated macrophages, Bcl-xL expression is independent of PI3K/Akt activation. Taken together, these results suggest that survival of macrophages is distinctly regulated during and after differentiation. Our results also suggest new, potential therapeutic targets to modulate differentiation and survival of this cell type.

Legumain expression, activity and secretion are increased during monocyte-to-macrophage differentiation and inhibited by atorvastatin.

Macrophages express several lysosomal cysteine proteases such as cathepsins and legumain. In this study, we assessed the expression, activity and secretion of legumain in cellular models of monocytes/macrophages. Macrophages were derived from M-CSF- or GM-CSF/IFNγ-stimulated human primary monocytes (M2 and M1, respectively), PMA-treated human THP-1 cells, or murine RAW264.7 macrophages. In both primary monocytes and THP-1 cells, monocyte-to-macrophage differentiation caused highly increased cellular expression and activity of legumain. Also, secretion of legumain from macrophages, but not from monocytes, was observed. Notably, M2 macrophages expressed significantly higher levels of active legumain than M1 macrophages, which are not previously reported. Legumain mRNA has been shown to be down-regulated in monocytes isolated from patients treated with the HMG-CoA reductase inhibitor atorvastatin. Interestingly, in our study, the active legumain produced by M2 macrophages was found to be inhibited by atorvastatin, which was reflected in aberrant cellular expression and processing.

Abstract 655: 4-Phenylbutyrate in Drinking Water Reduced Atherosclerotic Lesion Growth in ApoE-/- Mice by Modifying Monocyte-Macrophage Differentiation

Cardiovascular disease and atherothrombosis account for the majority of deaths worldwide. Plaque rupture, thrombus formation, and vessel occlusion contribute to the acute clinical manifestations of atherothrombosis. Previously we have shown that the unfolded protein response (UPR) has an important role in monocyte-macrophage differentiation. Furthermore, macrophage infiltration, lipid accumulation and foam cell formation are key events in atherogenesis. Endoplasmic reticulum (ER) stress-dependent apoptosis also contributes to atherosclerotic lesion growth and plaque necrosis. The aim of the current study was to examine whether 4-phenylbutyrate (4-PBA), a small chemical chaperone that alleviates ER stress, confers protection against atherosclerosis when provided via drinking water. To investigate the ability of 4-PBA to affect atherosclerotic lesion growth, at 12 weeks of age female ApoE-/- mice on chow diet were provided with 4-PBA (1 - 2.5 g/kg/d) in their drinking water for five weeks, ad libitum. Following treatment, mice were sacrificed, their tissues collected and atherosclerotic lesions examined by immunohistochemistry. Early atherosclerotic lesion area was significantly reduced in ApoE-/- mice provided 4-PBA-treated water from 12 - 17 weeks of age, relative to controls (0.6 x105 ± 0.1 μm2 versus 1.1 x105 ± 0.1 μm2, respectively, P < 0.05). We hypothesized that 4-PBA drinking water decreased lesion size by a mechanism involving reduced ER stress and monocyte-macrophage differentiation. In support of this mechanism, preliminary data indicates 4-PBA inhibited monocyte-macrophage differentiation, as measured by mRNA expression of the macrophage marker MSR-1 (0.5 ± 0.2 fold-change vs. control) and cell adherence (37 ± 5% vs. control) in PMA-treated THP-1 cells. In summary, these findings provide evidence that 4-PBA-treated drinking water reduced atherosclerotic lesion size in a mouse model of atherosclerosis. We hypothesize that 4-PBA acts in part through its ability to reduce ER stress and modulate monocyte/macrophage differentiation and function. Additional studies are in progress to further examine the mechanisms by which 4-PBA affects atherogenesis.

Activation of the NFκB Pathway Enhances AhR Expression in Intestinal Caco-2 Cells

Recent data suggest that apart from its well-known role in the regulation of xenobiotic metabolizing enzymes, AhR is also involved in inflammation. However, the influence of inflammation on AhR expression remains unknown. Here, we demonstrated that proinflammatory conditions induced by either PMA or IL-1β enhance AhR expression in Caco-2 cells. This was associated with an increase in AhR promoter activity. By means of directed mutagenesis experiments and the use of proteasome inhibitors, we demonstrated that inflammation-induced AhR expression involved the NFκB pathway but not AP-1. Moreover, conditioned media from PMA-treated Caco-2 cells were also able to induce AhR expression, and this induction was repressed by anti-IL-1β blocking antibodies. Similar results were obtained with conditioned media from PMA-treated THP-1 cells. Taken together, these data suggest that AhR could be involved in vivo in an inflammatory loop. AhR was recently suspected to be implicated in inflammatory bowel disease. Our results support this hypothesis and suggest that AhR could be a new target for inflammatory bowel disease patient management.

Down-Regulation of the Oncogene PTTG1 via the KLF6 Tumor Suppressor during Induction of Myeloid Differentiation

The aberrant expression of proto-oncogenes is involved in processes that are responsible for cellular proliferation and the inhibition of myeloid differentiation in acute myeloid leukemia (AML). Pituitary Tumor-Transforming gene 1 (PTTG1), an oncogenic transcription factor, is abundantly expressed in various human cancers and hematopoietic malignancies. However, its expression in normal leukocytes and most normal tissues is very low or undetectable. The mechanism by which PTTG1 overexpression modifies myeloid cell development and promotes leukemogenesis remain unclear. To investigate the mechanistic links between PTTG1 overexpression and leukemia cell differentiation, we utilized phorbol 12-myristate 13-acetate (PMA), a well-known agent that triggers monocyte/macrophage differentiation, to analyze the expression patterns of PTTG1 in PMA-induced myeloid differentiation. We found that PTTG1 is down-regulated at the transcriptional level in PMA-treated HL-60 and THP1 cells. In addition, we identified a binding site for a tumor suppressor protein, Kruppel-like factor 6 (KLF6), in the PTTG1 promoter. We found that KLF6 could directly bind and repress PTTG1 expression. In HL-60 and THP1 cells, KLF6 mRNA and protein levels are up-regulated with a concordant reduction of PTTG1 expression upon treatment with PMA. Furthermore, KLF6 knockdown by shRNA abolished the suppression of PTTG1 and reduced the activation of the differentiation marker CD11b in PMA-primed cells. The protein kinase C (PKC) inhibitor and the MAPK/ERK kinase (MEK) inhibitor significantly blocked the potentiation of PMA-mediated KLF6 induction and the down-regulation of PTTG1, indicating that PTTG1 is suppressed via the activation of PKC/ERK/KLF6 pathway. Our findings suggest that drugs that increase the KLF6 inhibition of PTTG1 may have a therapeutic application in AML treatment strategies.

Triptolide induces apoptosis of PMA-treated THP-1 cells through activation of caspases, inhibition of NF-κB and activation of MAPKs

Triptolide is known to be involved in many cellular events, such as those related to immunosuppressive and antitumor activity. We investigated whether triptolide mediates these effects through multiple mechanisms, including activation of cell cycle arrest and caspase-dependent pathways, as well as by blocking nuclear factor-κB (NF-κB) activation and by potentiating the activities of the mitogen-activated protein kinase (MAPK) pathway, in phorbol myristate acetate (PMA)-differentiated THP-1 cells. Triptolide significantly inhibited cell proliferation in a dose- and time-dependent manner and it increased the apoptotic fraction in the cell cycle and the number of apoptotic THP-1 cells. Exposure of the cells to triptolide also increased caspase-3 activity in these cells. Furthermore, co-treatment of cells with triptolide and the pan-caspase inhibitor, Z-VAD-FMK, or the caspase-3 inhibitor, Z-DEVE-FMK, increased THP-1 cell growth. Triptolide treatment resulted in a significant decrease in mRNA expression levels in genes encoding Bcl-2, cyclin D1, p27 and survivin and an increase in those encoding Bax and p21 in THP-1 cells. Triptolide not only inhibited NF-κB activation, but also activated p38 MAPK and MEK/ERK phosphorylation. These results show that triptolide inhibits the growth of THP-1 cells by inducing apoptosis through caspase activation and the mechanism involves NF-κB inhibition and the MAPK pathway.

Human IL10 Gene Repression by Rev-erbα Ameliorates Mycobacterium tuberculosis Clearance

Nuclear receptors modulate macrophage effector functions, which are imperative for clearance or survival of mycobacterial infection. The adopted orphan nuclear receptor Rev-erbα is a constitutive transcriptional repressor as it lacks AF2 domain and was earlier shown to be present in macrophages. In the present study, we highlight the differences in the relative subcellular localization of Rev-erbα in monocytes and macrophages. The nuclear localization of Rev-erbα in macrophages is subsequent to monocyte differentiation. Expression analysis of Rev-erbα elucidated it to be considerably more expressed in M1 phenotype in comparison with M2. Rev-erbα overexpression augments antimycobacterial properties of macrophage by keeping IL10 in a basal repressed state. Further, promoter analysis revealed that IL10 promoter harbors a Rev-erbα binding site exclusive to humans and higher order primates and not mouse, demonstrating a species barrier in its functionality. This direct gene repression is mediated by recruitment of co-repressors NCoR and HDAC3. In addition, our data elucidate that its overexpression reduced the survival of intracellular pathogen Mycobacterium tuberculosis by enhancing phagosome lysosome maturation, an event resulting from IL10 repression. Thus, these findings suggest that Rev-erbα bestows protection against mycobacterial infection by direct gene repression of IL10 and thus provide a novel target in modulating macrophage microbicidal properties.