PMA-stimulated Cells Research Articles

Simultaneous effects of hydrostatic pressure and dexamethasone release from electrospun fibers on inflammation-induced chondrocytes

Osteoarthritis (OA) is one of the major causes of disability worldwide. The characteristics of OA are increasing cell death, production of inflammatory cytokines, and changes in the function of the cell nucleus in the synthesis of destructive enzymes. Tissue engineering is a promising and ongoing process to suppress inflammation and rebuild the lost tissue. The aim of this research study is to investigate the simultaneous effect of hydrostatic pressure and dexamethasone (DEX) on immortalized human chondrocytes cell lines (C28/I2). To achieve this, first, dexamethasone (DEX) loaded electrospun poly(lactic acid) (PLA) fibers were prepared via electrospinning. Then, the release profile of DEX was evaluated under hydrostatic pressure (HP) with different magnitudes and frequencies. Based on the release profile of DEX from electrospun fibers and the physiological hydrostatic pressure of the cartilage, one of the applied regimes has been selected. Afterwards, C28/I2 was cultured on electrospun poly(lactic acid) (PLA) and DEX loaded PLA electrospun fiber and was grown in static and HP situation.After 7 days of exposure phorbol-12-myristate-13-acetate (PMA) to cultured cells on DEX loaded PLA scaffolds under hydrostatic pressure condition, cell viability and collagen deposition increased 3.63 and 1.85 times and nitric oxide production decreased 0.242 times in comparison to cultured PMA-stimulated cells on DEX loaded PLA. Moreover, through real time-PCR analysis, it was demonstrated that pairing HP and DEX led to reduced inducible nitric-oxide synthase (iNOS) and matrix metalloproteinase-13 (MMP-13) expressions by 0.01 and 0.041 times in comparison to static groups. However, expression of collagen type II did not enhance in the coupled system of HP and DEX. In conclusion, the presence of HP increased the DEX release from the nanofibrous scaffold and improved its ability as a drug depot scaffold for outstanding reduction of inflammation factors.

Blockade Effects of Anti-Interferon- (IFN-) γ Autoantibodies on IFN-γ-Regulated Antimicrobial Immunity.

The interferon- (IFN-) γ expression is elicited in response to microbial infections and activates immune surveillance by antimicrobial immune elements to induce microbial killing. Patients with adult-onset immunodeficiency who suffer from recurrent infections with microbes, particularly nontuberculous mycobacteria (NTM), commonly display genetic defects in IFN-γ signaling as well as the generation of anti-IFN-γ autoantibodies (autoAbs). Because IFN-γ is an activator of macrophage differentiation and a proinflammatory activator of innate immunity, the blockade effects of the autoAbs present in NTM patient serum on IFN-γ are hypothesized to regulate the antimicrobial function of macrophages. In the presence of patient serum, IFN-γ-induced type 1 macrophage (M1) differentiation was inhibited in PMA-stimulated human monocytic THP-1 cells. Treatment with patient serum significantly blocked the production of proinflammatory factors, including cytokines/chemokines and reactive oxygen/nitrogen species, by M1 macrophages. Importantly, IFN-γ-facilitated phagocytosis and degradation of heat-killed mycobacterium were decreased by cotreatment with patient serum. These results show the blockade activity of anti-IFN-γ autoAbs on IFN-γ-mediated antimicrobial immunity in macrophages.

In vitro activation and maturation of human mononuclear phagocytes by stimulation with liposomes coated with a neoglycolipid containing α1-3, α1-6-mannotriose.

In this study, we assessed the potential of liposomes coated with a neoglycolipid containing α1-3,α1-6-mannotriose residues (Man3-DPPE; Manα1-6(Manα1-3)Manitol-DPPE) for in vitro activation and maturation of human mononuclear phagocytes. In response to treatment with Man3-DPPE-coated liposomes (Man3-OMLs), PMA-stimulated human THP-1 cells showed enhanced expression of CD40, CD80 and HLA-DR and secreted significant levels of IL-12p40. Among various linkages of Man2-DPPE-coated liposomes, only liposomes coated with Manα1-6Manitol-DPPE (α1-6Man2-DPPE) induced these cellular responses similarly to Man3-OML treatment. Liposomes coated with Manα1-6(Manα1-3)Manα1-6(Manα1-3)Manitol-DPPE (Man5-DPPE) failed to activate the cells. These results suggest that an unsubstituted α1-6Man branch bound to a mannitol unit at the reducing end in Man3-DPPE is required for in vitro activation of human mononuclear phagocytes. Man3-OML-induced IL-12p40 production was not inhibited by BAY11-7082, an inhibitor of the MyD88-dependent signaling network, suggesting that TLRs are not involved in activation of human mononuclear phagocytes by Man3-OMLs. Stimulation of inflammatory monocytes or monocyte-derived dendritic cells (moDCs) with Man3-OMLs also induced enhanced expression of co-stimulatory molecules, HLA-DR, and CCR7, and IL-12p40 production from both types of cells. In response to Man3-OML treatment, moDCs but not inflammatory monocytes produced bioactive IL-12p70, which was enhanced by CD40 ligation. Thus, Man3-OMLs can activate naïve human mononuclear phagocytes and lead human moDCs to a fully matured status in vitro to elicit CTLs and a Th1 response without addition of inflammatory cytokines or TLR agonists.

Serotonin amide with anti‐inflammatory and wound healing properties

Some serotonin amides, as N‐behenoyl‐5‐hydroxytryptamine (C22:0‐5‐HT), are present in the surface wax of coffee beans and few studies show their possible biological activity. Thus, the aim of this work was to evaluate the anti‐inflammatory effect of C20:0‐5‐HT in the model of carrageenan‐induced inflammation into the subcutaneous air pouch (SAP), production of reactive oxygen species (ROS) by PMA‐stimulated leukocytes and MTT cell viability assay healing effect on the contraction model of the lesion in diabetic mice. C22:0‐5‐HT was synthesized by the group of Professor Claudia Rezende, Chemistry Institute, UFRJ. Female Webster mice (20–25g; n= 6–8) received oral administration of C22:0‐5‐HT (0.1, 1, 3 or 10 mg/kg) and were evaluated in model of SAP. Mice received carrageenan injection into the SAP and after 24 h were euthanized and the exsudate from SAP were collected for measurement of cell count. For the evaluation of ROS, leukocytes from the SAP were collected and incubated with C22:0‐5‐HT (0.1, 1 or 3 μM) for 1 h. Cells were incubated with 10 nM phorbol myristate acetate and 2 mM 2′‐7′‐dichlorodihidrofluoescein diacetate. The results were expressed as DCF‐DA fluorescence. Cell viability was measured by the MTT assay using RAW 264.7 mouse macrophages. For the analysis of healing activity female Webster mice (30–35g, n=10–12) received alloxan (65 mg/kg, iv) and after 7 days were anesthetized with ketamine and xylazine. The dorse was trichotomized and an area of 10 mm in diameter was exposed. The animals received topical administration (3 mg/animal) for 14 days and photos were taken on days 0, 3, 7, 10 and 14 to follow the retraction of the lesion. The image was processed in ImageJ program and the results are presented as the mean ± standard deviation of the wound area and expressed as arbitrary units (AU). Statistical significance between groups was determined by ANOVA followed by Bonferroni's test (*p<0.05) and the number of the animal research ethical committee is DFBCICB015‐04/16. C22:0‐5‐HT showed anti‐inflammatory activity in the three higher doses tested with reduction on cell migration (vehicle= 60.8 ± 8 × 106 cells/mL versus 61.7 ± 7 × 106 cells/mL, 31.2 ± 6* × 106 cells/mL, 37.1 ± 7* × 106 cells/mL, and 30.4 ± 6* × 106 cells/mL), with the doses of 0.1, 1, 3, and 10 mg/kg, respectively. It was also observed reduction in levels of ROS by PMA‐stimulated leukocytes in all concentrations tested (vehicle= 197,237.4 ± 8 fluorescence versus 98,852.1 ± 9* fluorescence, 87,951.3 ± 8.* fluorescence, 77,932.1 ± 7* fluorescence) and did not reduce the cell viability. Was observed improvement in healing of the treated mice on days 10 and 14 after injury. Naïve: day 0 = 40.1 ± 3.1 AU; 3 = 29.2 ± 5.1 AU; 7 = 13.1 ± 3.4 AU; 10 = 6.1 ± 2.2 AU; 14 = 3.2 ± 1.1 AU; Diabetic mice: day 0 = 41.3 ± 5.1 AU; 3 = 30.2 ± 5.4 AU; 7 = 20.1 ± 4.4 AU; 10 = 13 ± 3.5 AU; 14 = 7.2 ± 2 AU; Diabetic mice C22:0‐5‐HT‐treated (3 mg/animal) day 0 = 37.4 ± 6 AU; 3 = 30.9 ± 4 AU; 7 = 14.1 ± 3 AU; 10 = 2.7 ± 0.9* AU; 14 = 0.3 ± 0.1* AU. The results demonstrate that C22:0‐5‐HT produces anti‐inflammatory and healing effect. The mechanism(s) still under investigation.Support or Funding InformationThis study was financed in part by the CNPq and FAPERJ.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Epimagnolin A inhibits IL-6 production by inhibiting p38/NF-κB and AP-1 signaling pathways in PMA-stimulated THP-1 cells.

Epimagnolin A is a lignan obtained from the flower buds of Magnolia fargesii, which is traditionally used in Asian medicine for treating headache and nasal congestion. A herbal compound fargesin obtained from M. fargesii, has exerted anti-inflammatory effects in human monocytic THP-1 cells in the previous study. The anti-inflammatory effects of epimagnolin A, however, have been not elucidated yet. In this study, it was demonstrated that epimagnolin A reduced phorbol-12-myristate-13-acetate (PMA)-induced IL-6 promoter activity and IL-6 production in human monocytic THP-1 cells. Furthermore, it was investigated the modulating effects of epimagnolin A on mitogen-activated protein kinase, nuclear factor-kappa B (NF-κB), and activator protein 1 (AP-1) activities. Phosphorylation of p38 and nuclear translocation of p50 and c-Jun were down-regulated by epimagnolin A in the PMA-stimulated THP-1 cell. The results revealed that epimagnolin A attenuated the binding affinity of NF-κB and AP-1 transcription factors to IL-6 promoter and IL-6 production through p38/NF-kB and AP-1 signaling pathways in the PMA-stimulated THP-1 cells. These results suggest that epimagnolin A can be a useful drug for the treatment of inflammatory diseases.

Mannan-Binding Lectin Suppresses Peptidoglycan-Induced TLR2 Activation and Inflammatory Responses.

Peptidoglycan (PGN), as the major components of the bacterial cell wall, is known to cause excessive proinflammatory cytokine production. Toll-like receptor 2 (TLR2) is abundantly expressed on immune cells and has been shown to be involved in PGN-induced signaling. Although more and more evidences have indicated that PGN is recognized by TLR2, the role of TLR2 PGN recognition is controversial. Mannan-binding lectin (MBL), a plasma C-type lectin, plays a key role in innate immunity. More and more evidences show that MBL could suppress the amplification of inflammatory signals. Whether MBL can alter PGN-elicited cellular responses through TLR2 in macrophages is still unknown, and possible mechanism underlying it should be investigated. In this study, we found that MBL significantly attenuated PGN-induced inflammatory cytokine production, including TNF-α and IL-6, in PMA-stimulated THP-1 cells at both mRNA and protein levels. The expression of TLR2 was strongly induced by PGN stimulation. Furthermore, the administration of TLR2-neutralized antibody effectively suppressed PGN-induced TNF-α and IL-6 expression. These results supplied the evidence that PGN from Saccharomyces cerevisiae could be recognized by TLR2. In addition, we also found that MBL decreased PGN-induced TLR2 expression and suppressed TLR2-mediated downstream signaling, including the phosphorylation of IκBα, nuclear translocation of NF-κBp65, and phosphorylation of MAPK p38 and ERK1/2. Administration of MBL alone did not have an effect on the expression of TLR2. Finally, our data showed that PGN-mediated immune responses were more severely suppressed by preincubation with MBL and indicated that MBL can combine with both TLR2 and PGN to block the inflammation cytokine expression induced by PGN. All these data suggest that MBL could downregulate inflammation by modulating PGN/TLR2 signaling pathways. This study supports an important role for MBL in immune regulation and signaling pathways involved in inflammatory responses.

Targeted Disruption of the Cohesin Subunit STAG2 Leads to Loss of Insulation and Inappropriate Gene Activation in Response to Differentiation Signals

Cohesin is a multiprotein complex that mediates sister chromatid cohesion during S phase of the cell cycle, but it also plays crucial roles in genome organization and transcription in interphase. Cohesin's role in regulation of the genome helps determine lineage identity and differentiation of hematopoietic cells (Viny et al., JEM, 2015; Mullenders et al., JEM, 2015; Mazumdar et al., Cell stem cell, 2015; Galeev et al., Cell reports, 2016). Mitotic cohesin is composed of RAD21, SMC1A, SMC3 and STAG1/STAG2 subunits. Mutations in the subunits of cohesin complex are present in significant proportion of myeloid malignancies (Thota et al., Blood 2014). In particular, the frequency of cohesin subunit mutation is as high as ~50% in Down-syndrome associated megakaryoblastic leukemia subtype (Yoshida et al., Nature Genetics, 2013).To further understand cohesin's role in gene regulation in a leukemia context, we used CRISPR-CAS9 to engineer a leukemia associated STAG2 mutation, R614*, into K562 leukemia cells to generate isogenic model lines. Homozygous mutant K562 cells showed complete loss of STAG2 expression. We also obtained STAG2 R614* heterozygous cell clones that showed partial loss of STAG2 expression (STAG2 is on the X chromosome, and the wild type allele is mosiacally silenced in heterozygotes).Phenotype analyses of STAG2 mutant K562 cells showed that homozygous mutants had altered morphology and had acquired adherent properties. Homozygous mutant cells activated expression of the stem cell marker, KIT, and showed a reduction in the neutrophil differentiation marker, CD15.We used ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) to examine changes in chromatin accessibility, and RNA sequencing to determine changes in gene expression in STAG2 mutants. More than 5,000 genes were dysregulated in STAG2 homozygous mutants, accompanied by profound changes in chromatin accessibility with gain and loss of accessibility represented equally. Loss of accessibility was observed at insulators that recruit CCCTC-binding factor (CTCF), indicating potential loss of insulation at chromatin boundaries. Conversely, gain of chromatin accessibility was observed at regions that are normally repressed in K562, and this was accompanied by activation of transcription factors such as ERG and FLI1. Gain of accessibility was also observed at myeloid-associated super enhancers of the RUNX1 gene.We hypothesized that loss of STAG2 leads to an inability to constrain transcriptional programs upon differentiation of leukemia cells. To test this hypothesis, we stimulated the STAG2 mutant and parental K562 cells with phorbol myristate acetate (PMA) to induce megakaryocyte differentiation. PMA-stimulated STAG2 homozygous mutant cells showed a striking precocious spike in RUNX1 expression that was associated with enhanced transcription from its proximal P2 promoter. A similar precocious spike was observed in transcription of ERG. Interestingly, the spike in transcription of these genes was not observed at 48 hours, indicating that the ectopic transcription of RUNX1 and ERG is confined to early response to stimulation. The BET inhibitor JQ1, was previously shown to block enhancer activity (Bhagwat et al., Cell reports, 2016). Treatment of STAG2 mutant cells with JQ1 dampened ectopic RUNX1 P2 expression and led to a complete loss of RUNX1 P1 and ERG activation during PMA stimulation. These results suggest that ectopic RUNX1 expression in response to STAG2 deficiency is enhancer-driven. Furthermore, JQ1 treatment caused a 2-fold decrease in KIT levels in STAG2-deficient cells, indicating a reversal of immature properties in these cells.In summary, our results suggest that STAG2 mutation results in loss of chromatin insulation, leading to inappropriate activation of hematopoietic enhancers. Loss of constraints on enhancer-driven transcription could in turn underlie the aberrant gene expression in STAG2-deficient cells exposed to differentiation cues. DisclosuresNo relevant conflicts of interest to declare.

PMA treated THP-1-derived-IL-6 promotes EMT of SW48 through STAT3/ERK-dependent activation of Wnt/β-catenin signaling pathway

Colon cancer is one of the most common digestive malignant tumors that leads to high mortality worldwide, and metastasis is the primary cause of cancer-related death. It is well accepted that the epithelial-mesenchymal transition (EMT) plays a key role in the process of metastasis. As a cytokine that macrophage secretes, IL-6 is involved in the progression of tumors, including the invasion and metastasis via kinds of signaling pathways. However, the mechanism of interactions between IL-6, macrophage, EMT and colon cancer is not fully understood. Increased CD68+ macrophages and IL-6 level were found in colon tumor as compared to normal colon tissue. Metastatic lymph node showed even more CD68+ macrophages and higher IL-6 level than the primary tumor. These results suggested that macrophages and IL-6 play an important role in EMT of colon cancer. In order to investigate the effect of macrophage and IL-6 on EMT of colon cancer, we cultured human colon carcinoma cell line SW48 with conditioned medium (CM) from PMA-stimulated monocyte THP-1 cells and tested for IL-6 dependent EMT pathways. Wound healing assay and Transwell assay were used to analyze cell migration and invasion. Results showed that CM-treated SW48 cells increased IL-6 production and displayed elevated capacity of migration and invasion compared to untreated cells. Increased expressions of EMT markers (N-cadherin, Vimentin and β-catenin) and decreased expression of EMT marker(E-cadherin) were found in CM-treated SW48 cells by Western Blot. The addition of an anti-IL-6 antibody significantly inhibited the increase of EMT markers (Vimentin and β-catenin) as well as cell migration and invasion, suggesting that IL-6 played a critical role in promoting EMT of CM-treated SW48 cells. In addition, we found that the levels of p-STAT3 and p-ERK increased in CM-treated SW48 compared to untreated cells, which can be reversed by AG490, an inhibitor of JAK. In the meantime, the suppression of JAK-associated signaling pathways caused a decrease of β-catenin. In summary, our study suggested that macrophage-induced IL-6 promotes migration and invasion of colon cancer cell via Wnt/β-catenin pathway in STAT3/ERK-dependent way.

Rapid on-site dual optical system to measure specific reactive oxygen species (O2-• and OCl-) in a tiny droplet of whole blood.

Oxidative stress has been implicated in various disorders and controlling it would be important for healthy life. We have developed a new optical system for easily and accurately measuring oxidative stress in whole blood. It is optimized for simultaneously detecting reactive oxygen species (ROS) and highly reactive ROS (hROS), elicited mostly by white blood cells in a few microliters of blood. Results obtained by using this system show at least four important findings. 1) chemiluminescence of MCLA was confirmed to be attributable to O2-•. 2) PMA-stimulated cells released O2-• longer and more slowly than fMLP-stimulated ones. 3) fluorescence produced by APF oxidation was confirmed to be attributable to hROS, mostly OCl-, produced by myeloperoxidase. 4) the generation of OCl- was found to be a slower process than the O2-• generation. We also conducted pilot studies of oxidative stress in healthy volunteers.

Lipoxin A4 and its analog suppress hepatocarcinoma cell epithelial-mesenchymal transition, migration and metastasis via regulating integrin-linked kinase axis

Epithelial-mesenchymal Transition (EMT) and migration play an important role in tumor progression, and lipoxin (LX), the ‘stop signal’ for inflammation, has been studied in basic research for its anti-inflammatory or inflammatory pro-resolving properties. Here, in the in vitro experiment, we showed that LXA4 could inhibit the EMT and migration in phorbol myristate acetate (PMA) or activated conditioned medium (ACM)-stimulated Hep3B cells by downregulation of integrin-linked kinase (ILK), a pseudokinase in cytoplasm and these effects were via inhibiting the phosphorylation of Akt and GSK3β. Morover, LXA4 could not affect the EMT and migration of PMA-stimulated Hep3B cells by knockdown of ILK. In the in vivo experiment, BML-111 (the analog of LXA4) could inhibit the EMT and metastasis of hepatocarcinoma cells. We also demonstrated that ILK siRNA inhibited phosphorylation of downstream signaling targets Akt and GSK3β, decreased expression of MMP-2 and MMP-9. These results showed that LXA4 could be a possible candidate for liver cancer therapy, and blocking ILK axis would be an effective drug target.

Synthesis of a Cleaved Form of Osteopontin by THP-1 Cells and Its Alteration by Phorbol 12-Myristate 13-Acetate and BCG Infection.

The protease-cleaved osteopontin (OPN) was proposed to enhance the migration of memory T cells to granulomas in tuberculosis. Various forms of OPN were identified in human monocytic THP-1 cells stimulated by phorbol 12-myristate 13-acetate (PMA). Antibodies O-17, 10A16 and 34E3, which recognize N-terminus, the C-half, and thrombin-cleaved site of OPN, respectively, all detected distinct bands on Western blots following PMA stimulation. Bands corresponding to 18 and 30 kD were detected by antibodies 34E3 and 10A16, indicating that OPN cleavage occurred by endogenous proteases in the PMA-stimulated THP-1 cells. In immune-fluorescence (IF) assay, 34E3 positive signals were detected in intracellular space of non-infected and bacillus Calmette-Guérin (BCG)-infected cells; however, 10A16 positive signals were confirmed in extracellular area in PMA-stimulated cells followed by BCG infection. Small amounts of full-length (FL) and thrombin-cleaved (Tr) OPN were detected by ELISA in the supernatants of non-PMA-stimulated cells, and increased levels of all forms, including undefined (Ud) OPN, in PMA-stimulated cells. ELISA showed a decrease in OPN synthesis during BCG infection. To our knowledge, this is the first report of OPN cleavage in THP-1 macrophages after PMA stimulation, and of enhanced cleavage induced by BCG infection.

Water-Soluble Fluorescent Probe with Dual Mitochondria/Lysosome Targetability for Selective Superoxide Detection in Live Cells and in Zebrafish Embryos.

A novel water-soluble fluorescein-based fluorescent probe for superoxide detection was developed. The probe is fairly stable under neutral and acidic conditions. It can be used to detect superoxide both in solution with the detection limit of 2.2 μM and in living cells. Cell imaging experiments indicated that such a probe displayed good cell penetration and O2•- could be detected with PMA-stimulated HepG2 cells in both mitochondria and lysosome. Such a probe is the first dual mitochondria- and lysosome- targetable fluorescent chemodosimeter. Additionally, O2•- in intact live zebrafish embryos was successfully visualized under PMA-stimulated conditions, and the possible detection mechanism was studied as well.

Gelatin promotes cell aggregation and pro-inflammatory cytokine production in PMA-stimulated U937 cells by augmenting endocytosis-autophagy pathway.

Gelatin, denatured collagen, temporarily exists in tissues and may well be pathophysiologically involved in tissue remodeling, inflammation or tissue damage. The present study is aimed to investigate possible biological roles of gelatin by examining its effects on monocyte-like histiocytic lymphoma cell line U937. Once stimulated by phorbol 12-myristate 13-acetate (PMA), U937 cells differentiate into macrophage-like cells, changing from non-adherent to adherent cells with extended pseudopodia. Here we pre-treated the cell dishes with gelatin solution for cell culture. Interestingly, we found that PMA-stimulated U937 cells formed multicellular aggregates on gelatin-coated dishes, accompanying NF-κB-mediated production of pro-inflammatory cytokines, whereas cell aggregation was not detected on non-coated dishes. Moreover, differentiated U937 cells on gelatin-coated dishes showed increased autophagy level and endocytosis. Surprisingly, formation of multicellular aggregates and pro-inflammatory cytokine production were both attenuated by either down-regulation of autophagy with inhibitors, such as 3-methyladenine (3MA) or chloroquine (CQ), or repression of endocytosis with siRNA targeting Endo180. Moreover, autophagy was inhibited by si-Endo180, and endocytosis was suppressed by 3MA, suggesting a positive feedback loop between autophagy and endocytosis. The results revealed that gelatin-coating induced differentiated U937 cells to form cell aggregates and promote NF-κB-mediated pro-inflammatory cytokine production at least partially through an endocytosis-autophagy pathway.

Phorbol ester (PMA)-treated U937 cells cultured on type I collagen-coated dish express a lower production of pro-inflammatory cytokines through lowered ROS levels in parallel with cell aggregate formation

The present study is aimed to investigate the effect of collagen I on U937 cells, human monocyte-like histiocytic lymphoma cell line. Differentiation of U937 cells was induced by phorbol ester (PMA) treatment. The cells were cultured on the collagen I-coated plate. PMA-stimulated U937 cells formed multicellular aggregates on collagen I-coated surface, whereas PMA-unstimulated cells kept themselves away off each other. Moreover, the levels of reactive oxygen species (ROS) and productions of pro-inflammatory cytokines such as IL-1β, TNFα and PGE2, pro-inflammatory mediator, were down-regulated in differentiated U937 cells cultured on collagen I-coated dishes. However, collagen I did not influence the capacity of E. coli phagocytosis. Cell aggregation as well as the down-regulation of IL-1β, TNFα and PGE2 caused by the culture on collagen I-coated surface were suppressed by ROS donor, tert-butylhydroperoxide (tBHP). The sizes of cell aggregates became bigger in differentiated U937 cells by treatment with ROS scavengers such as N-acetylcysteine (NAC), superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH). In conclusion, collagen I-coated culture induces the differentiated U937 cells to form cell aggregates and decreases the production of pro-inflammatory cytokines through down-regulating ROS generation.

BODIPY labeled Cellulose Nanocrystals (CNCs) Induces Il-1β secretion and Antioxidant Response in a Mitochondrial ROS-independent Manner

Cellulose nanocrystals (CNCs) are rod-like nanomaterials that can be easily obtained via acidic hydrolysis of native cellulose. There has been a growing interest in modifying CNCs for biomedical purposes, including bioimaging. This emerging interest in CNCs lead us to synthesize a functionalized CNCs with a green-fluorescent BODIPY. Since needle-like molecules as well as fluorophores may induce reactive oxygen species (ROS), and these aspects are associated with NLRP3 inflammasome activation, the goal this study was to investigate whether CNCs-BIODIPY increases mitochondrial ROS leading to increases in the intracellular antioxidant defense and inflammatory response. Mouse macrophage-like cells (J774A.1) and PMA-stimulated human monocyte cells (THP-1) were treated with different concentrations of CNCs-BIODIPY, in presence or absence of LPS. After treatment, J774A.1 cells were harvested and intracellular levels of inflammatory proteins (NLRP3 and IL-1β) and the antioxidant enzymes (PrxSO3, Catalase and SOD1) were analyzed by western blotting in cell lysates. Mature IL-1β levels were analyzed by ELISA in cell supernatants and lysates from both cell lines and mitochondrial ROS by MitoSox in J774A.1 cells. The results indicated that levels of NLRP3 and pro-IL-1 β were increased in non LPS-stimulated cells as well as the antioxidant enzymes catalase and oxidized peroxiredoxins, PrxSO3. However, no significant changes in mitochondrial ROS production was found, in comparison to control conditions. Overall, our results revealed that CNCs-BIOPIDY increased antioxidant enzymes and NLRP3 inflammasome pathway suggesting that it may not be suitable to systemic biomedical applications in the tested concentrations.

단핵구세포주 THP-1의 대식세포로의 분화 및 활성화에서 CO의 억제 효과

Carbon monoxide (CO), a reaction product of cytoprotective enzyme heme oxygenase-1 (HO-1), is a gaseous messenger with anti-proliferative, anti-apoptotic, and anti-inflammatory actions in many cell types. Here, we investigated the role of CO on the process of monocyte differentiation induced by phorbol 12-myristate 13-acetate (PMA) in human monocytic THP-1 cells. CORM-2 (tricarbonyldichlororuthenium (II) dimer, Ru₂Cl₄ (CO) 6 ), a CO-releasing compound, decreased a marked cell adherence with a slight reduction of proliferation in monocytic THP-1 cells treated with PMA. And, CORM-2 significantly inhibited expression of differentiation markers such as CD14, CD11b plus CD18 (macrophage-1 antigen, Mac-1 or complement receptor 3, CR3) and phagocytosis of carboxylate-modified red fluorescent latex beads, in PMA-stimulated THP-1 cells. For the further experiments, differentiation of PMA-treated cells was enhanced after the initial 2 days stimulus by removing the PMA-containing media then incubating the cells in fresh media for a another 4 days. And, we observed the secretion of inflammatory cytokines and phagocytosis in differentiated macrophages. Treatment with CORM-2 significantly abolished the secretion of IL-6, TNF-α and phagocytosis using fluorescence-conjugated E. coli (K-12 strain) bioparticles in lipopolysaccharide (LPS)-stimulated differentiated macrophages. In conclusion, these results suggest that CO inhibits the differentiation of monocytic THP-1 cells as well as the activation of differentiated macrophages.

Effects of heme degradation products on reactivation of latent HIV-1.

Human immunodeficiency virus (HIV-1) infection can be currently controlled by combined antiretroviral therapy, but a sterilizing cure is not achievable as this therapy does not target persistent HIV-1 in latent reservoirs. Therefore, different latency reversal agents are intensively explored in various models. We have previously observed that heme arginate, a drug approved for human use, reveals a strong synergism with PKC inducers in reactivation of the latent provirus. Heme is physiologically decomposed by heme oxygenases into 3 degradation products: iron (Fe2+), carbon monoxide (CO) and biliverdin which is further converted to bilirubin by biliverdin reductase. In this paper, we have studied the effects of individual heme-degradation products on latent HIV-1 reactivation in ACH-2 cells harboring integrated HIV-1 provirus and in H12 clone of Jurkat cells harboring HIV-minivirus expressing EGFP. We employed addition of ascorbate to generate Fe2+, resulting in increased expression of both HIV-1 p24 Ag and EGFP in PMA-stimulated ACH-2 and H12 cells, respectively, as characterized on RNA and protein levels. On the other hand, addition of a CO-donor or bilirubin decreased the p24 expression. The reactivation of latent HIV-1 by iron or heme arginate was inhibited by antioxidant N-acetyl cysteine, or by an iron chelator desferrioxamine, suggesting that the effects were mediated by iron- or heme-induced redox stress. Finally, we demonstrated the stimulatory effects of heme arginate and PMA on HIV-1 expression in peripheral blood mononuclear cells of HIV-infected patients cultured ex vivo. These results may constitute a new direction in the latent HIV-1 reactivation and therapy.

Mometasone Furoate Suppresses PMA-Induced MUC-5AC and MUC-2 Production in Human Airway Epithelial Cells.

BackgroundMucus hypersecretion from airway epithelium is a characteristic feature of airway inflammatory diseases. Tumor necrosis factor α (TNF-α) regulates mucin synthesis. Glucocorticoids including mometasone fuorate (MF) have been used to attenuate airway inflammation. However, effects of MF on mucin production have not been reported.MethodsEffects of MF and budesonide (BUD) on the phorbol-12-myristate-13-acetate (PMA)–induction of mucin and TNF-α in human airway epithelial cells (NCI-H292) were investigated in the present study. Confluent NCI-H292 cells were pretreated with PMA (200 nM) for 2 hours. Subsequently, the cells were stimulated with MF (1–500 ng/mL) or BUD (21.5 ng/mL) for 8 hours. Dexamethasone (1 µg/mL) was used as the positive control. Real-time polymerase chain reaction was used to determine MUC2 and MUC5AC mRNA levels. The level of total mucin, MUC2, MUC5AC, and TNF-α in culture supernatants were measured using enzyme-linked immunosorbent assay.ResultsMF and BUD significantly suppressed MUC2 and MUC5AC gene expression in PMA-stimulated NCI-H292 cells. The inhibitory effects of the two steroid drugs were also observed in the production of total mucin, MUC2 and MUC5AC proteins, and TNF-α.ConclusionOur findings demonstrated that MF and BUD attenuated mucin and TNF-α production in PMA-induced human airway epithelial cells.

Atorvastatin suppresses NLRP3 inflammasome activation via TLR4/MyD88/NF-κB signaling in PMA-stimulated THP-1 monocytes.

Increasing evidence shows that NLRP3 inflammasome is closely associated with the progression of atherosclerosis. The purpose of the present study was to evaluate the effects of atorvastatin on NLRP3 inflammasome in PMA-stimulated THP-1 cells and explore its underlying mechanism. Human monocytic THP-1 cells were pretreated with atorvastatin for 1h and then induced by PMA for 48h. Total protein was collected for real-time PCR and Western blot analysis. Cytokine IL-1β release was detected by ELISA assay. And the NF-κB p65 translocation was detected by cellular NF-κB translocation kit. It was shown that atorvastatin significantly reduced the expression of NLRP3, the cleavage of caspase-1 and IL-1β in PMA-induced THP-1 cells. Moreover, Bay (a NF-κB inhibitor) treatment greatly suppressed the expression of NLRP3, the cleavage of caspase-1 and IL-1β in PMA-induced THP-1 cells, suggesting that the activation of NF-κB pathway takes part in regulating the expression of NLRP3 inflammasome. In addition, atorvastatin markedly inhibited the up-regulation of toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88) and the activation of nuclear factor kappa b (NF-κB) in PMA-stimulated THP-1 cells. Atorvastatin exerts an anti-inflammatory effect by inhibiting NLRP3 inflammasome through suppressing TLR4/MyD88/NF-κB pathway in PMA-induced THP-1 monocytes.

A role for multiple estrogen receptors in immune regulation of common carp

Estrogens are important for bi-directional neuroendocrine-immune interaction. They act via nuclear estrogen receptors (ERα and ERβ) and/or G-protein coupled receptor - GPR30.We found expression of ERα, ERβ and GPR30 in carp lymphoid tissues and head kidney monocytes/macrophages, neutrophils and lymphocytes. Interestingly, ERβ is also expressed in some head kidney lymphocytes but not in naive PBLs. Immune stimulation altered the cell type specific profile of expression of these receptors, which depends on both activation and maturation stage.This implies direct leukocyte responsiveness to estrogen stimulation and therefore in vitro effects of 17β-estradiol (E2) on reactive oxygen species (ROS) production in monocytes/macrophages were determined. Short-time incubation with E2 increased ROS production in PMA-stimulated cells. Results comply with mediation by GPR30, partially functioning via phosphoinositide 3-kinase activation.These results furthermore demonstrate that neuroendocrine-immune communication via estrogen receptors is evolutionary conserved.