Monocytic U937 Research Articles

Palmitoylation is required for TNF-R1 signaling

BackgroundBinding of tumor necrosis factor (TNF) to TNF-receptor 1 (TNF-R1) can induce either cell survival or cell death. The selection between these diametrically opposed effects depends on the subcellular location of TNF-R1: plasma membrane retention leads to survival, while endocytosis leads to cell death. How the respective TNF-R1 associated signaling complexes are recruited to the distinct subcellular location is not known. Here, we identify palmitoylation of TNF-R1 as a molecular mechanism to achieve signal diversification.MethodsHuman monocytic U937 cells were analyzed. Palmitoylated proteins were enriched by acyl resin assisted capture (AcylRAC) and analyzed by western blot and mass spectrometry. Palmitoylation of TNF-R1 was validated by metabolic labeling. TNF induced depalmitoylation and involvement of APT2 was analyzed by enzyme activity assays, pharmacological inhibition and shRNA mediated knock-down. TNF-R1 palmitoylation site analysis was done by mutated TNF-R1 expression in TNF-R1 knock-out cells. Apoptosis (nuclear DNA fragmentation, caspase 3 assays), NF-κB activation and TNF-R1 internalization were used as biological readouts.ResultsWe identify dynamic S-palmitoylation as a new mechanism that controls selective TNF signaling. TNF-R1 itself is constitutively palmitoylated and depalmitoylated upon ligand binding. We identified the palmitoyl thioesterase APT2 to be involved in TNF-R1 depalmitoylation and TNF induced NF-κB activation. Mutation of the putative palmitoylation site C248 interferes with TNF-R1 localization to the plasma membrane and thus, proper signal transduction.ConclusionsOur results introduce palmitoylation as a new layer of dynamic regulation of TNF-R1 induced signal transduction at a very early step of the TNF induced signaling cascade. Understanding the underlying mechanism may allow novel therapeutic options for disease treatment in future.

Ovarian cancer stem cells and macrophages reciprocally interact through the WNT pathway to promote pro-tumoral and malignant phenotypes in 3D engineered microenvironments

BackgroundInnate immune cells such as macrophages are abundantly present within malignant ascites, where they share the microenvironment with ovarian cancer stem cells (CSC).MethodsTo mimic this malignant ascites microenvironment, we created a hanging-drop hetero-spheroid model to bring CSCs and macrophages in close association. Within these hetero-spheroids, CD68+ macrophages (derived from U937 or peripheral blood monocytes) make up ~ 20% of the population, while the rest are ovarian cancer cells and ovarian cancer stem cells (derived from the high grade serous ovarian cancer cell line, OVCAR3).ResultsOur results indicate that CSCs drive the upregulation of M2 macrophage marker CD206 within hetero-spheroids, compared to bulk ovarian cancer cells, implying an inherently more immuno-suppressive program. Moreover, an increased maintenance of elevated aldehyde dehydrogenase (ALDH) activity is noted within hetero-spheroids that include pre-polarized CD206+ M2 macrophages, implying a reciprocal interaction that drives pro-tumoral activation as well as CSC self-renewal. Consistent with enriched CSCs, we also observe increased levels of pro-tumoral IL-10 and IL-6 cytokines in the CSC/M2-macrophage hetero-spheroids. CSC/M2-macrophage hetero-spheroids are also less sensitive to the chemotherapeutic agent carboplatin and are subsequently more invasive in transwell assays. Using inhibitors of WNT secretion in both CSCs and macrophages, we found that CSC-derived WNT ligands drove CD206+ M2 macrophage activation, and that, conversely, macrophage-derived WNT ligands enriched ALDH+ cells within the CSC compartment of hetero-spheroids. Upon examination of specific WNT ligand expression within the monocyte-derived macrophage system, we observed a significant elevation in gene expression for WNT5B. In CSCs co-cultured with macrophages within hetero-spheroids, increases in several WNT ligands were observed, and this increase was significantly inhibited when WNT5B was knocked down in macrophages.ConclusionsOur data implies that macrophage- initiated WNT signaling could play a significant role in the maintenance of stemness, and the resulting phenotypes of chemoresistance and invasiveness. Our results indicate paracrine WNT activation during CSC/M2 macrophages interaction constitutes a positive feedback loop that likely contributes to the more aggressive phenotype, which makes the WNT pathway a potential target to reduce the CSC and M2 macrophage compartments in the tumor microenvironment.

The Establishment and Validation of the Human U937 Cell Line as a Cellular Model to Screen Immunomodulatory Agents Regulating Cytokine Release Induced by Influenza Virus Infection.

Severe influenza infections are often associated with the excessive induction of pro-inflammatory cytokines, which is also referred to as “cytokine storms”. Several studies have shown that cytokine storms are directly associated with influenza-induced fatal acute lung injury and acute respiratory distress syndrome. Due to the narrow administration window, current antiviral therapies are often inadequate. The efforts to use immunomodulatory agents alone or in combination with antiviral agents in the treatment of influenza in animal models have resulted in the achievement of protective effects accompanied with reduced cytokine production. Currently, there are no immunomodulatory drugs for influenza available for clinical use. Animal models, despite being ideal to study the anti-inflammatory responses to influenza virus infection, are very costly and time-consuming. Therefore, there is an urgent need to establish fast and economical screening methods using cell-based models to screen and develop novel immunomodulatory agents. In this study, we screened seven human cell lines and found that the human monocytic cell U937 supports the replication of different subtypes of influenza viruses as well as the production of the important pro-inflammatory cytokines and was selected to develop the cell-based model. The U937 cell model was validated by testing a panel of known antiviral and immunomodulatory agents and screening a drug library consisting of 1280 compounds comprised mostly of FDA-approved drugs. We demonstrated that the U937 cell model is robust and suitable for the high-throughput screening of immunomodulators and antivirals against influenza infection.Electronic supplementary materialThe online version of this article (10.1007/s12250-019-00145-w) contains supplementary material, which is available to authorized users.

Propofol attenuates monocyte-endothelial adhesion via modulating connexin43 expression in monocytes

AimsMonocyte-endothelial adhesion is considered to be the primary initiator of inflammatory vascular diseases, such as atherosclerosis. Connexin 43 (Cx43) has been reported to play an important part in this process, however, the underlying mechanisms are not fully understood. Intravenous anesthetics, propofol is commonly used in the perioperative period and in the intensive care unit, and considered to have good anti-inflammatory and antioxidant effects. Thus, we speculate that propofol could influence monocyte-endothelial adhesion, and explore whether its possible mechanism is relative with Cx43 expression in U937 monocytes influencing cell adhesion of U937 monocytes to human umbilical vein endothelial cells (HUVEC). Main methodsCx43-siRNAs or pc-DNA-Cx43 were used to alter Cx43 expression in U937 monocytes. Propofol was given as pretreatments to U937 monocytes. Then, cell adhesion, ZO-1, LFA-1, VLA-4, COX and MCP-1 were determined. PI3K/AKT/NF-κB signaling pathway was explored to clarify the possible mechanism. Key findingsAlternation of Cx43 expression affects cell adhesion and adhesion molecules significantly, such as ZO-1, LFA-1, VLA-4, COX-2 and MCP-1, the mechanism of which is relative with Cx43 influencing the activation of PI3K/AKT/NF-κB signaling pathway. Preconditioning with propofol at its clinically relevant anesthesia concentration attenuates cell adhesion. Propofol not only decreases Cx43 expression in U937 monocytes, but also depresses the activation of PI3K/AKT/NF-κB signaling pathway. SignificanceModulation Cx43 expression in U937 monocytes could affect cell adhesion via regulating the activation of PI3K/AKT/NF-κB signaling pathway. Propofol attenuates cell adhesion via inhibiting Cx43 and its downstream signaling pathway of PI3K/AKT/NF-κB.

Decrease in fat de novo synthesis and chemokine ligand expression in non-alcoholic fatty liver disease caused by inhibition of mixed lineage kinase domain-like pseudokinase.

Receptor-interacting serine/threonine kinase 3 and mixed lineage kinase domain-like pseudokinase (MLKL) have gained attention as apoptosis alternate cell death signaling molecules. We aimed to evaluate the role of MLKL in non-alcoholic fatty liver disease (NAFLD). Hepatic tissue MLKL expression was compared between NAFLD patients and healthy controls. High-fat diet was fed to wild-type and MLKL-knockout (KO) mice for 12weeks. Brown adipose fat tissue was measured by [18 F]-fluorodeoxyglucose positron emission tomography. Energy expenditure was measured by indirect calorimetry. Anti-MLKL effects were also evaluated in in vitro setting using U937 and HepG2 cells. Hepatic tissue MLKL expression increased in NAFLD patients compared with healthy controls. MLKL expression increased according to the degree of steatosis, ballooning, and inflammation. High-fat diet-fed MLKL-KO mice displayed decreased alanine aminotransferase, triglycerides, liver weight, NAFLD activity score (6.3 vs 3.5, P<0.001), steatosis score (3.0 vs 1.8, P<0.001), inflammation, and ballooning degeneration compared with wild-type mice. SREBP1c, fatty acid synthase, and SCD-1 expressions decreased in MLKL-KO mice. Adipose tissue F4/80-positive crown-like structures were also reduced in MLKL-KO mice. HepG2 cells treated with necrosulfonamide (an MLKL inhibitor) showed reduced Nile red staining and reduced SREBP1c and SCD-1 expressions. Stimulation of necroptosis using lipopolysaccharide+caspase inhibitor (zVAD) increased CXCL1/2 expressions in U937 monocyte cells. Lipopolysaccharide+zVAD-induced increased expressions of CXCL1/2 were reduced with necrosulfonamide treatment. Mixed lineage kinase domain-like pseudokinase inhibition has protective effects in non-alcoholic steatohepatitis by decreasing hepatic de novo fat synthesis and chemokine (C-X-C motif) ligand expressions.

Ponatinib Protects Mice From Lethal Influenza Infection by Suppressing Cytokine Storm.

Excessive inflammation associated with the uncontrolled release of pro-inflammatory cytokines is the main cause of death from influenza virus infection. Previous studies have indicated that inhibition of interferon gamma-induced protein 10 (IP-10), interleukin-8 (IL-8), monocyte chemoattractant protein 1 (MCP-1), or their cognate receptors has beneficial effects. Here, by using monocytic U937 cells that capable of secreting the three important cytokines during influenza A virus infection, we measured the inhibitory activities on the production of three cytokines of six anti-inflammatory compounds reported in other models of inflammation. We found that ponatinib had a highly inhibitory effect on the production of all three cytokines. We tested ponatinib in a mouse influenza model to assess its therapeutic effects with different doses and administration times and found that the delayed administration of ponatinib was protective against lethal influenza A virus infection without reducing virus titers. Therefore, we suggest that ponatinib may serve as a new immunomodulator in the treatment of influenza.

Effects of enhancer of Zeste homolog 2 inhibitor GSK126 on the cell proliferation and apoptosis of acute leukemia and lymphoma cells in vitro

Objective To investigate the effects of a novel enhancer of Zeste homolog 2 (EZH2) inhibitor GSK126 in vitro on the cell proliferation and apoptosis in acute leukemia and lymphoma cells. Methods CCK-8 assay was used to measure the effects of GSK126 with different concentrations and time on the cell proliferation in human T-cell acute lymphoblastic leukemia (T-ALL) CEM cell line, acute monocytic leukemia U937 cell line and Burkitt lymphoma Raji cell line. Annexin V/PI double staining method was used to determine the effects of GSK126 on the cell apoptosis. The effect of GSK126 on the mRNA levels of EZH2, bcl-2 and bcl-xL were measured by using quantitative polymerase chain reaction (qPCR). Results After the function for 24 h, 48 h and 72 h, compared to the negative control group (0 μmol/L), 5, 10, 15, 20, 25 μmol/L GSK126 treatment showed a significant dose-and time-dependent cell proliferation suppression in CEM cells; 5, 10, 15, 20 μmol/L GSK126 treatment showed a significant dose-and time-dependent cell proliferation suppression in U937 cells; 5, 10, 15, 20, 25, 30 μmol/L GSK126 treatment showed a significant time-dependent suppression in Raji cells (all P < 0.05). The 48 h 50% inhibitory concentration (IC50) of GSK126 on CEM, U937, Raji cells was (13.46±0.83), (11.65±1.02), (15.00±0.19) μmol/L, respectively. GSK126 treatment with the doses of 8, 12 and 16 μmol/L promoted the apoptosis of CEM and U937 cells compared with the negative control group (0 μmol/L), and there were statistical differences in the apoptosis rate (F = 167.995, P < 0.01; F = 158.400, P < 0.01). In Raji cells, only 16 μmol/L GSK126 treatment had a higher cell apoptosis rate compared with the control group (t = 47.998, P < 0.05). Furthermore, 8, 12 and 16 μmol/L GSK126 treatment suppressed the expressions of EZH2 mRNA in CEM, U937 and Raji cells compared with the control group (F = 82.035, P < 0.01; F = 252.712, P < 0.01; F = 690.536, P < 0.01), and the expressions of bcl-2 and bcl-xL mRNA (bcl-2: F = 1 900.525, P < 0.01; F = 431.324, P < 0.01; F = 216.184, P < 0.01; bcl-xL: F = 256.751, P < 0.01; F = 147.019, P < 0.01; F = 209.325, P < 0.01). Conclusion EZH2 plays an important role in the occurrence and development of acute leukemia and lymphoma. GSK126 as a high selective inhibitor of EZH2 might be a potential new drug in treatment of hematological malignancies. Key words: Hematologic neoplasms; Cell proliferation; Apoptosis; Enhancer of Zeste homolog 2; GSK126

Low-density lipoprotein as an opsonin promoting the phagocytosis of Pseudomonas aeruginosa by U937 cells.

Low-density lipoprotein (LDL) was recently reported to be an opsonin, enhancing the phagocytosis of group A Streptococcus (GAS) by human monocytic leukemia U937 cells due to the binding of LDL to some GAS strains. We postulated that LDL might also promote the opsonophagocytosis of Pseudomonas aeruginosa by U937 cells since this bacterium interacts with LDL. In this study, P. aeruginosa (CMCC10104), U937 cells, and human LDL were used in phagocytosis assays to test our hypothesis. Escherichia coli strain BL21, which does not interact with LDL, was used as a negative control. Colony counting and fluorescence microscopy were used to determine the bacterial quantity in the opsonophagocytosis assays. After incubation of U937 cells and P. aeruginosa with LDL (100 µg/ml) for 15 and 30 min, phagocytosis was observed to be increased by 22.71% and 32.90%, respectively, compared to that seen in the LDL-free group. However, LDL did not increase the phagocytosis of E. coli by U937 cells. In addition, we identified CD36 as a major opsonin receptor on U937 cells, since an anti-CD36 monoclonal antibody, but not an anti-CD4 monoclonal antibody, almost completely abolished the opsonophagocytosis of P. aeruginosa by U937 cells.

SLPI Inhibits ATP-Mediated Maturation of IL-1β in Human Monocytic Leukocytes: A Novel Function of an Old Player

Interleukin-1β (IL-1β) is a potent, pro-inflammatory cytokine of the innate immune system that plays an essential role in host defense against infection. However, elevated circulating levels of IL-1β can cause life-threatening systemic inflammation. Hence, mechanisms controlling IL-1β maturation and release are of outstanding clinical interest. Secretory leukocyte protease inhibitor (SLPI), in addition to its well-described anti-protease function, controls the expression of several pro-inflammatory cytokines on the transcriptional level. In the present study, we tested the potential involvement of SLPI in the control of ATP-induced, inflammasome-dependent IL-1β maturation and release. We demonstrated that SLPI dose-dependently inhibits the ATP-mediated inflammasome activation and IL-1β release in human monocytic cells, without affecting the induction of pro-IL-1β mRNA by LPS. In contrast, the ATP-independent IL-1β release induced by the pore forming bacterial toxin nigericin is not impaired, and SLPI does not directly modulate the ion channel function of the human P2X7 receptor heterologously expressed in Xenopus laevis oocytes. In human monocytic U937 cells, however, SLPI efficiently inhibits ATP-induced ion-currents. Using specific inhibitors and siRNA, we demonstrate that SLPI activates the calcium-independent phospholipase A2β (iPLA2β) and leads to the release of a low molecular mass factor that mediates the inhibition of IL-1β release. Signaling involves nicotinic acetylcholine receptor subunits α7, α9, α10, and Src kinase activation and results in an inhibition of ATP-induced caspase-1 activation. In conclusion, we propose a novel anti-inflammatory mechanism induced by SLPI, which inhibits the ATP-dependent maturation and secretion of IL-1β. This novel signaling pathway might lead to development of therapies that are urgently needed for the prevention and treatment of systemic inflammation.

Genistein protects against DSS-induced colitis by inhibiting NLRP3 inflammasome via TGR5-cAMP signaling

NLRP3 inflammasome has been reported to be associated with inflammatory bowel disease including colitis due to its potential ability to induce IL-1β secretion. Emerging studies have demonstrated that Genistein, a major isoflavone, has potential anti-inflammatory effects in murine model colitis. However, its anti-inflammatory mechanism remains unclear. The effects of Genistein in dextran sulfate sodium (DSS)-induced murine colitis via targeting NLRP3 inflammasome was investigated in this study. Also, the mechanisms of protective action of Genistein in DSS-induced colitis may relate to TGR5 signaling. Genistein treatment not only remarkably attenuated loss of body weight and shortening of colon length but also significantly reduced inflammatory cells infiltration and pro-inflammatory mediator production in serum and colon. Moreover, Genistein treatment down-regulated production of caspase-1 and IL-1β and increased intracellular cAMP level, which were similar to the treatment for INT-777, a semi-synthetic TGR5 agonist, in phorbol myristate acetate (PMA)-differentiated monocytic THP-1 cells and U937 cells. These protective effects of Genistein might be attributed by ubiquination of NLRP3 which was induced due to interaction of cAMP with NLRP3. Furthermore, the effects of Genistein on NLRP3 inflammasome disappeared in TGR5-silenced U937 cells. In conclusion, our study unveils that Genistein was able to inhibit NLRP3 inflammasome via TGR5-cAMP signaling in macrophages. It therefore might be a potential effective drug for inflammatory bowel diseases.

Antiatherogenic effects of liraglutide in hyperglycemic apolipoprotein E-null mice via AMP-activated protein kinase-independent mechanisms.

Glucagon-like peptide-1 receptor agonists (GLP-1RAs) exert potent glucose-lowering effects without increasing risks for hypoglycemia and weight gain. Preclinical studies have demonstrated direct antiatherogenic effects of GLP-1RAs in normoglycemic animal models; however, the underlying mechanisms in hyperglycemic conditions have not been fully clarified. Here we aimed to elucidate the role of AMP-activated protein kinase (AMPK) in antiatherogenic effects of GLP-1RAs in hyperglycemic mice. Streptozotocin-induced hyperglycemic apolipoprotein E-null mice were treated with vehicle, low-dose liraglutide (17 nmol·kg-1·day-1), or high-dose liraglutide (107 nmol·kg-1·day-1) in experiment 1 and the AMPK inhibitor dorsomorphin, dorsomorphin + low-dose liraglutide, or dorsomorphin + high-dose liraglutide in experiment 2. Four weeks after treatment, aortas were collected to assess atherosclerosis. In experiment 1, metabolic parameters were similar among the groups. Assessment of atherosclerosis revealed that high-dose liraglutide treatments reduced lipid deposition on the aortic surface and plaque volume and intraplaque macrophage accumulation at the aortic sinus. In experiment 2, liraglutide-induced AMPK phosphorylation in the aorta was abolished by dorsomorphin; however, the antiatherogenic effects of high-dose liraglutide were preserved. In cultured human umbilical vein endothelial cells, liraglutide suppressed tumor necrosis factor-induced expression of proatherogenic molecules; these effects were maintained under small interfering RNA-mediated knockdown of AMPKα1 and in the presence of dorsomorphin. Conversely, in human monocytic U937 cells, the anti-inflammatory effects of liraglutide were abolished by dorsomorphin. In conclusion, liraglutide exerted AMPK-independent antiatherogenic effects in hyperlipidemic mice with streptozotocin-induced hyperglycemia, with the possible involvement of AMPK-independent suppression of proatherogenic molecules in vascular endothelial cells.

Triglycerides increase mRNA Expression of Pro-inflammatory Cytokines Via the iNOS in Jurkat T lymphocyte and U937 Monocyte Cell Lines

Triglycerides (TG) are one of the triggers of chronic inflammatory lesions in the blood vessels. In the key factors in the development of inflammatory diseases, Pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-)α and interleukin-1 beta (IL-1β) contribute to the development of inflammatory lesions by recruiting other immune cells in the inflamed area or causing cell necrotic death. In this study, I investigated the effect of Jurkat T lymphocytes and U937 monocytes involved in vascular inflammation development on the expression of TNF-α and IL-1β on exposure to TGs. In Jurkat cells, mRNA expression of TNF-α is increased by exposure to TGs. However, the expression levels of TNF-α and IL-1β were increased by TGs in U937 cells. To investigate whether inducible nitric oxide synthase (iNOS) is involved in the increase of expression of TNF-α and IL-1β by TGs, treatment of W1400 (an iNOS inhibitor) resulted in recovery of expression level both TNF- α and IL-1β. Based on the present study, it was confirmed that the expression of TNF-α and IL-1β in monocytes and T lymphocytes. This increased cytokines contribute to development of vascular inflammatory lesions. In addition, iNOS is involved in the increase of TNF-α and IL-1β expression by TGs.

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

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

Long-Lasting Anti-Inflammatory Activity of Human Microfragmented Adipose Tissue.

Over the last few years, human microfragmented adipose tissue (MFAT), containing significant levels of mesenchymal stromal cells (MSCs) and obtained from fat lipoaspirate (LP) through a minimal manipulation in a closed system device, has been successfully used in aesthetic medicine as well as in orthopedic and general surgery. Interestingly, in orthopedic diseases, this ready-to-use adipose tissue cell derivative seems to have a prolonged time efficacy even upon a single shot injection into osteoarthritic tissues. Here, we investigated the long-term survival and content of MSCs as well the anti-inflammatory activity of LP and its derived MFAT in vitro, with the aim to better understand a possible in vivo mechanism of action. MFAT and LP specimens from 17 human donors were investigated side by side. During a long-term culture in serum-free medium, we found that the total cell number as well the MSC content in MFAT decreased more slowly if compared to those from LP specimens. The analysis of cytokines and growth factors secreted into the conditioned medium (CM) was similar in MFAT and LP during the first week of culture, but the total amount of cytokines secreted by LP decreased much more rapidly than those produced by MFAT during prolonged culture (up to 28 days). Similarly, the addition of MFAT-CM recovered at early (3-7 days) and late stage (14-28 days) of culture strongly inhibited inflammatory function of U937 monocyte cell line, whereas the anti-inflammatory activity of LP-CM was drastically reduced after only 7 days of culture. We conclude that MFAT is an effective preparation with a long-lasting anti-inflammatory activity probably mediated by a long-term survival of their MSC content that releases a combination of cytokines that affect several mechanisms involved in inflammation processes.

Protein Disulfide Isomerase Inhibitor Suppresses Viral Replication and Production during Antibody-Dependent Enhancement of Dengue Virus Infection in Human Monocytic Cells.

One of several mechanisms that leads to the development of dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) is called antibody-dependent enhancement (ADE). Monocytes can be infected by the ADE phenomenon, which occurs in dengue secondary infection. This study aimed to investigate the proteins involved in ADE of DENV infection in the human monocytic cell line U937. The phosphoproteins were used to perform and analyze for protein expression using mass spectrometry (GeLC-MS/MS). The differential phosphoproteins revealed 1131 altered proteins compared between isotype- and DENV-specific antibody-treated monocytes. The altered proteins revealed 558 upregulated proteins and 573 downregulated proteins. Protein disulfide isomerase (PDI), which is an enzyme that had a high-ranking fold change and that catalyzes the formation, breakage, and rearrangement of disulfide bonds within a protein molecule, was selected for further study. PDI was found to be important for dengue virus infectivity during the ADE model. The effect of PDI inhibition was also shown to be involved in the early stage of life cycle by time-of-drug-addition assay. These results suggest that PDI is important for protein translation and virion assembly of dengue virus during infection in human monocytes, and it may play a significant role as a chaperone to stabilize dengue protein synthesis.

Semispecific TPPII inhibitor Ala-Ala-Phe-chloromethylketone (AAF-cmk) displays cytotoxic activity by induction of apoptosis, autophagy and protein aggregation in U937 cells.

The main component of extralysosomal proteolysis is the ubiquitin-proteasome system (UPS), which is supplemented by tripeptidyl peptidase II (TPPII). That system is a target for anticancer strategies by using proteasome inhibitors. Data from several studies on leukemic cells share evidence for the beneficial and potential role of TPPII in cell survivability. Therefore, the aim of this work was to analyze the effect of AAF-cmk, a membrane permeable semi-specific TPPII inhibitor, on human monocytic leukemic cells U937 for translational research. We studied the viability of U937 cells incubated with AAF-cmk using tetrazolium salt reduction assay (MTT) and apoptosis induction by assessing caspase activation by Western blotting and Annexin V binding assays. Transmission electron microscopy (TEM), a gold standard for apoptosis and autophagy detection, was used to assess the ultrastructure of U937 cells. Incubation of cells with AAF-cmk reduced their viability and induced apoptosis by intrinsic pathway. In groups treated with AAF-cmk, activation of caspases 9 and 3 was observed and caspase inhibition by zVDA restored cell viability. TEM revealed the presence of ultrastructural features of apoptosis and authophagy. Moreover, we identified two types of protein aggregates. The first one was found in close proximity to the endoplasmic reticulum (ER) and corresponds to Aggresome-Like Structure (ALIS); however, the second novel type of aggregate was not related to ER elements, but rather to free cytosolic ribosomes. This type did not correspond to the aggresome neither in localization nor the structure, thus we referred these aggregates as ALiSNER (Aggresome-Like Structure Not Associated With the ER). Our results provide novel and important findings about the role of TPPII in protein homeostasis and cell survival. Since semispecific TPPII inhibitor AAF-cmk displays cytotoxic activity against leukemic U937 cells in vitro it can be considered as a potential anticancer agent.

HIV-1-derived exosomal microRNAs miR88 and miR99 promote the release of cytokines from human alveolar macrophages by binding to TLR8

The human monocytic cell line U937 and human alveolar macrophages were used as in vitro models to explore the role of miR88 and miR99 in the chronic abnormal activation of the body caused by human immunodeficiency virus (HIV). The functions and underlying mechanisms of miR88 and miR99 were studied by real-time quantitative polymerase chain reaction, transwell, and chromatin immunoprecipitation (ChIP) assays. HIV-1-infected cells released miR88 and miR99 into the extracellular space through exosomes, and miR88 and miR99 promoted the release of tumor necrosis factor alpha (TNFα), interleukin (IL)-6, and IL-12 by activating inflammatory factors, such as TLR8, on the surface of macrophages. HIV-derived microRNAs miR88 and miR99 performed these functions by binding to TLR8 and stimulating the release of pro-inflammatory factors from macrophages, such as TNFα, IL-6, and IL-12; these factors may be involved in chronic abnormal immune activation induced by HIV infection.

Monocyte phagocytosis of malaria β-haematin in the presence of artemisinin, amodiaquine, chloroquine, doxycycline, primaquine, pyrimethamine and quinine

The intraerythrocytic malaria parasite digests haemoglobin to provide amino acids for metabolism and releases toxic haem that is sequestered into haemozoin, a non-toxic, insoluble, crystalline pigment. Following erythrocyte rupture, haemozoin is released into circulation and phagocytosed by monocytes. Phagocytosed haemozoin and antimalarial drugs have both been reported to modulate monocyte functions. This study determined the effects of therapeutic concentrations of seven antimalarial drugs; amodiaquine, artemisinin, chloroquine, doxycycline, primaquine, pyrimethamine and quinine, on the phagocytosis of β-haematin (synthetic haemozoin) by two monocytic cell lines, J774A.1 and U937, and human peripheral blood mononuclear cells. A novel spectrophotometric method based on the absorbance (O.D 400 nm) of alkali/SDS treated monocytes containing β-haematin was developed to complement counting phagocytosis with microscopy. The method has potential use for the large scale screening of monocyte phagocytic activity. Artemisinin, quinine, primaquine and pyrimethamine activated β-haematin phagocytosis by 12% or more, whereas amodiaquine, chloroquine and doxycyline inhibited β-haematin phagocytosis. In contrast, antimalarial drugs had minimal inhibitory effects on the phagocytosis of latex beads with only quinine resulting in more than 20% inhibition. Antimalarial drugs appear to alter monocyte phagocytic activity which has implications for the treatment, pathogenicity and adjunct therapies for malaria.

Extracellular Vesicle-Mimetic Ghost Nanovesicles for Delivering Anti-Inflammatory Drugs to Mitigate Gram-Negative Bacterial Outer Membrane Vesicle-Induced Systemic Inflammatory Response Syndrome.

Sepsis is one of the major causes of death in hospital patients and is represented by systemic inflammatory response syndrome (SIRS) associated with infection. Gram-negative bacteria including Escherichia coli can provoke sepsis by stimulating the immune systems. Outer membrane vesicles (OMVs), nanosized vesicular structures derived from Gram-negative bacteria, contain several pathogen-associated molecular patterns, and are demonstrated to mediate SIRS. Here, extracellular vesicle-mimetic ghost nanovesicles loaded with dexamethasone, an anti-inflammatory drug, are developed using alkaline solution, sonication, and buoyant density gradient ultracentrifugation. These ghost nanovesicles have comparable physical features with naturally released extracellular vesicles but have 200-fold higher production yields than extracellular vesicles. Importantly, these ghost nanovesicles are devoid of potentially unwanted luminal cargos, including cytosolic proteins and nucleic acids. By maintaining the same topology as the parental cells, these dexamethasone-loaded ghost nanovesicles derived from human U937 monocytes reduce the release of interleukin-8 from OMV-treated endothelial cells in vitro, and mitigate the symptoms of OMV-induced SIRS in vivo. This study sheds light on using extracellular vesicle-mimetic ghost nanovesicles to deliver therapeutics to treat diseases such as bacterial sepsis.

Inhibitory effects of vasostatin-1 against atherogenesis.

Vasostatin-1, a chromogranin A (CgA)-derived peptide (76 amino acids), is known to suppress vasoconstriction and angiogenesis. A recent study has shown that vasostatin-1 suppresses the adhesion of human U937 monocytes to human endothelial cells (HECs) via adhesion molecule down-regulation. The present study evaluated the expression of vasostatin-1 in human atherosclerotic lesions and its effects on inflammatory responses in HECs and human THP-1 monocyte-derived macrophages, macrophage foam cell formation, migration and proliferation of human aortic smooth muscle cells (HASMCs) and extracellular matrix (ECM) production by HASMCs, and atherogenesis in apolipoprotein E-deficient (ApoE-/-) mice. Vasostatin-1 was expressed around Monckeberg's medial calcific sclerosis in human radial arteries. Vasostatin-1 suppressed lipopolysaccharide (LPS)-induced up-regulation of monocyte chemotactic protein-1 (MCP-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin in HECs. Vasostatin-1 suppressed inflammatory M1 phenotype and LPS-induced interleukin-6 (IL-6) secretion via nuclear factor-κB (NF-κB) down-regulation in macrophages. Vasostatin-1 suppressed oxidized low-density lipoprotein (oxLDL)-induced foam cell formation associated with acyl-CoA:cholesterol acyltransferase-1 (ACAT-1) and CD36 down-regulation and ATP-binding cassette transporter A1 (ABCA1) up-regulation in macrophages. In HASMCs, vasostatin-1 suppressed angiotensin II (AngII)-induced migration and collagen-3 and fibronectin expression via decreasing ERK1/2 and p38 phosphorylation, but increased elastin expression and matrix metalloproteinase (MMP)-2 and MMP-9 activities via increasing Akt and JNK phosphorylation. Vasostatin-1 did not affect the proliferation and apoptosis in HASMCs. Four-week infusion of vasostatin-1 suppressed the development of aortic atherosclerotic lesions with reductions in intra-plaque inflammation, macrophage infiltration, and SMC content, and plasma glucose level in ApoE-/- mice. These results indicate the inhibitory effects of vasostatin-1 against atherogenesis. The present study provided the first evidence that vasostatin-1 may serve as a novel therapeutic target for atherosclerosis.