1'-Acetoxychavicol acetate inhibits NLRP3-dependent inflammasome activation via mitochondrial ROS suppression.

The nucleotide-binding oligomerization domain-like receptor (NLR) family pyrin domain containing (NLRP) 3 inflammasome is a multiprotein complex that triggers Caspase-1-mediated IL-1ß production and pyroptosis, and its dysregulation is associated with the pathogenesis of inflammatory diseases. 1'-Acetoxychavicol acetate (ACA) is a natural compound in the rhizome of tropical ginger Alpinia species with anti-microbial, anti-allergic and anti-cancer properties. In this study, we found that ACA suppressed NLRP3 inflammasome activation in mouse bone marrow-derived macrophages and human THP-1 monocytes. ACA inhibited Caspase-1 activation and IL-1ß production by NLRP3 agonists such as nigericin, monosodium urate (MSU) crystals, and ATP. Moreover, it suppressed oligomerization of the adapter molecule, apoptosis-associated speck-like protein containing a CARD (ASC), and Caspase-1-mediated cleavage of pyroptosis executor Gasdermin D. Mechanistically, ACA inhibited generation of mitochondrial reactive oxygen species (ROS) and prevented release of oxidized mitochondrial DNA, which trigger NLRP3 inflammasome activation. ACA also prevented NLRP3 inflammasome activation in vivo, as evidenced in the MSU crystal-induced peritonitis and dextran sodium sulfate-induced colitis mouse models accompanied by decreased Caspase-1 activation. Thus, ACA is a potent inhibitor of the NLRP3 inflammasome for prevention of NLRP3-associated inflammatory diseases.

Distinct Effects of Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus Cell Wall Component-Induced Inflammation on the Iron Metabolism of THP-1 Cells.

Macrophages are essential immune cells of the innate immune system. They participate in the development and regulation of inflammation. Macrophages play a fundamental role in fighting against bacterial infections by phagocytosis of bacteria, and they also have a specific role in immunomodulation by secreting pro-inflammatory cytokines. In bacterial infection, macrophages decrease the serum iron concentration by removing iron from the blood, acting as one of the most important regulatory cells of iron homeostasis. We examined whether the Gram-positive and Gram-negative cell wall components from various bacterial strains affect the cytokine production and iron transport, storage and utilization of THP-1 monocytes in different ways. We found that S. aureus lipoteichoic acid (LTA) was less effective in activating pro-inflammatory cytokine expression that may related to its effect on fractalkine production. LTA-treated cells increased iron uptake through divalent metal transporter-1, but did not elevate the expression of cytosolic and mitochondrial iron storage proteins, suggesting that the cells maintained iron efflux via the ferroportin iron exporter. E. coli and P. aeruginosa lipopolysaccharides (LPSs) acted similarly on THP-1 cells, but the rates of the alterations of the examined proteins were different. E. coli LPS was more effective in increasing the pro-inflammatory cytokine production, meanwhile it caused less dramatic alterations in iron metabolism. P. aeruginosa LPS-treated cells produced a smaller amount of pro-inflammatory cytokines, but caused remarkable elevation of both cytosolic and mitochondrial iron storage proteins and intracellular iron content compared to E. coli LPS. These results prove that LPS molecules from different bacterial sources alter diverse molecular mechanisms in macrophages that prepossess the outcome of the bacterial infection.

Platelet Microparticles Decrease Daunorubicin-Induced DNA Damage and Modulate Intrinsic Apoptosis in THP-1 Cells.

Platelets can modulate cancer through budding of platelet microparticles (PMPs) that can transfer a plethora of bioactive molecules to cancer cells upon internalization. In acute myelogenous leukemia (AML) this can induce chemoresistance, partially through a decrease in cell activity. Here we investigated if the internalization of PMPs protected the monocytic AML cell line, THP-1, from apoptosis by decreasing the initial cellular damage inflicted by treatment with daunorubicin, or via direct modulation of the apoptotic response. We examined whether PMPs could protect against apoptosis after treatment with a selection of inducers, primarily associated with either the intrinsic or the extrinsic apoptotic pathway, and protection was restricted to the agents targeting intrinsic apoptosis. Furthermore, levels of daunorubicin-induced DNA damage, assessed by measuring gH2AX, were reduced in both 2N and 4N cells after PMP co-incubation. Measuring different BCL2-family proteins before and after treatment with daunorubicin revealed that PMPs downregulated the pro-apoptotic PUMA protein. Thus, our findings indicated that PMPs may protect AML cells against apoptosis by reducing DNA damage both dependent and independent of cell cycle phase, and via direct modulation of the intrinsic apoptotic pathway by downregulating PUMA. These findings further support the clinical relevance of platelets and PMPs in AML.

Progranulin inhibits LPS-induced macrophage M1 polarization via NF-кB and MAPK pathways.

BACKGROUND: Macrophage M1 polarization plays a pivotal role in inflammatory diseases. Progranulin (PGRN) has potential anti-inflammation action, however, the effect of PGRN on macrophage M1 polarization has been poorly studied. Our study aimed to investigate the effect of PGRN on lipopolysaccharide (LPS)-induced macrophage M1 polarization and clarify the underlying mechanisms. METHODS: RAW264.7 cells were polarized to M1 macrophage by LPS with or without recombinant PGRN (rPGRN) and tumor necrosis factor alpha antibody (anti-TNF-α). A cell counting kit-8 assay (CCK-8), flow cytometry, Quantitative Real-Time PCR assay (q-PCR), Western blot assay and enzyme-linked immunosorbent assay (ELISA) were used to determine the effect of different treatments on cell proliferation, expression of surface phenotype marker and expressions and secretion of inflammatory cytokines. The activation of NF-κB/mitogen-activated protein kinase (MAPK) pathways and the nuclear translocation of NF-κB p65 were detected by Western blot and immunofluorescence respectively. THP-1 and primary bone marrow-derived monocytes (BMDMs) were also used to demonstrate effect of PGRN on expressions and secretion of inflammatory cytokines induced by LPS. RESULTS: In RAW264.7 cells, rPGRN at concentrations below 80 ng/ml significantly promoted cell proliferation in dose dependent fashion. rPGRN significantly inhibited LPS-induced change of phenotype (CD86/CD206 ratio) and function (tumor necrosis factor (TNF-α) and inducible nitric oxide synthase (iNOS) expressions). LPS-stimulated secretion of TNF-α and activated phosphorylation of IKKα/ß, IкBα, p65, JNK and p38 and the nucleus translocation of NF-кB p65 were also significantly downregulated by rPGRN. In addition, recombinant TNF-α (rTNF-α) significantly boosted TNF-α and iNOS expression vs the control group. Moreover, anti-TNF-α significantly inhibited LPS-induced TNF-α and iNOS expression. In THP-1 and BMDM cells, reversing effect of rPGRN on LPS-enhanced expressions of TNF-α and iNOS and secretion of TNF-α was further demonstrated. CONCLUSIONS: PGRN down-regulates LPS-induced macrophage M1 polarization in phenotype and function via NF-κB/MAPK signaling pathways.

Lysophosphatidylcholines and Chlorophyll-Derived Molecules from the Diatom Cylindrotheca closterium with Anti-Inflammatory Activity.

Microalgae have been shown to be excellent producers of lipids, pigments, carbohydrates, and a plethora of secondary metabolites with possible applications in the pharmacological, nutraceutical, and cosmeceutical sectors. Recently, various microalgal raw extracts have been found to have anti-inflammatory properties. In this study, we performed the fractionation of raw extracts of the diatom Cylindrotheca closterium, previously shown to have anti-inflammatory properties, obtaining five fractions. Fractions C and D were found to significantly inhibit tumor necrosis factor alpha (TNF-⍺) release in LPS-stimulated human monocyte THP-1 cells. A dereplication analysis of these two fractions allowed the identification of their main components. Our data suggest that lysophosphatidylcholines and a breakdown product of chlorophyll, pheophorbide a, were probably responsible for the observed anti-inflammatory activity. Pheophorbide a is known to have anti-inflammatory properties. We tested and confirmed the anti-inflammatory activity of 1-palmitoyl-sn-glycero-3-phosphocholine, the most abundant lysophosphatidylcholine found in fraction C. This study demonstrated the importance of proper dereplication of bioactive extracts and fractions before isolation of compounds is commenced.

Stimulation of THP-1 Macrophages with LPS Increased the Production of Osteopontin-Encapsulating Exosome.

Osteopontin (OPN) mediates bone remodeling and tissue debridement. The OPN protein is cleaved, but it is unclear how full-length (FL)-OPN or its cleaved form perform their biological activities in target cells. We, therefore, performed the molecular characterization of OPN in exosomes (Exo). The Exo were isolated from lipopolysaccharide (LPS)-stimulated phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1 macrophages. The Exo were also isolated from PMA-differentiated THP-1 macrophages. The Exo were identified using the qNano multiple analyzer (diameter 59-315 nm) and western blotting with a CD9 antibody. LPS-stimulated cells produced more particles than non-stimulated cells. The presence of the FL or the cleaved form of OPN was confirmed using western blot analysis. A mixture of FL and cleaved OPN was also measured using an ELISA system (Ud-OPN) and their presence in the Exo was confirmed. Ud/FL ratios became low after LPS stimulation, indicating the enhanced encapsulation of FL-OPN in the Exo by LPS. These findings suggest that LPS stimulation of human macrophages facilitates the synthesis of FL-OPN, which is cleaved in cells or the Exo after release. These findings indicate that Exo is a suitable vehicle to transfer OPN to the target cells.

A Dipeptidyl Peptidase-4 Inhibitor Inhibits Foam Cell Formation of Macrophages in Type 1 Diabetes via Suppression of CD36 and ACAT-1 Expression.

Dipeptidyl peptidase-4 (DPP-4) inhibitors have been reported to play a protective role against atherosclerosis in both animal models and patients with type 2 diabetes (T2D). However, since T2D is associated with dyslipidemia, hypertension and insulin resistance, part of which are ameliorated by DPP-4 inhibitors, it remains unclear whether DPP-4 inhibitors could have anti-atherosclerotic properties directly by attenuating the harmful effects of hyperglycemia. Therefore, we examined whether a DPP-4 inhibitor, teneligliptin, could suppress oxidized low-density lipoprotein (ox-LDL) uptake, foam cell formation, CD36 and acyl-coenzyme A: cholesterol acyltransferase-1 (ACAT-1) gene expression of macrophages isolated from streptozotocin-induced type 1 diabetes (T1D) mice and T1D patients as well as advanced glycation end product (AGE)-exposed mouse peritoneal macrophages and THP-1 cells. Foam cell formation, CD36 and ACAT-1 gene expression of macrophages derived from T1D mice or patients increased compared with those from non-diabetic controls, all of which were inhibited by 10 nmol/L teneligliptin. AGEs mimicked the effects of T1D; teneligliptin attenuated all the deleterious effects of AGEs in mouse macrophages and THP-1 cells. Our present findings suggest that teneligliptin may inhibit foam cell formation of macrophages in T1D via suppression of CD36 and ACAT-1 gene expression partly by attenuating the harmful effects of AGEs.

Unexpected mechanism of colitis amelioration by artesunate, a natural product from Artemisia annua L.

BACKGROUND: Artemisinin and its derivatives are known to exert immunosuppressive effects through modulating adaptive immunity. We investigated a novel role of artesunate in regulating innate immunity, including both macrophages (MΦ) and dendritic cells (DCs), which are known to involve in DSS-induced colitis. METHODS: Effects of artesunate on innate immunity were extensively evaluated, both in vivo using DSS-colitis model with WT and T cell-deficient RAG mice (RAG-/-) and in vitro using cell culture models, including in-depth analyses of MΦ/DC apoptosis and cytokine expression by flow cytometry, Western blot, or immunohistology. RESULTS: Unexpectedly, artesunate significantly ameliorated the DSS colitis of both WT and RAG1-/- mice with similar potency, suggesting a mechanism that involves primarily innate rather than adaptive immunity. In vivo mechanistic studies revealed that artesunate markedly induced apoptosis of lamina propria MΦs and DCs and suppressed mucosal TNF-α and IL-12p70 in DSS-colitis. In vitro, artesunate potently induced a dose- and time-dependent apoptosis of murine bone marrow-derived DCs and human THP-1 MΦs, through the caspases-9-mediated intrinsic pathway. Artesunate significantly decreased the secretion of IL-12p40/70 by DCs and TNF-α by MΦs. Furthermore, a combination of artesunate with an immunomodulator (methotrexate/triptolide/azathioprine) exhibited superior potency in promoting apoptosis of MΦs than any individual drug alone. CONCLUSIONS: The immunomodulatory mechanism of artesunate in colitis involves a novel and potent induction of the intrinsic apoptosis pathway of proliferating MΦs and DCs and suppression of IL-12 and TNF-α. Artemisinin and its derivatives are promising new therapeutic alternatives for IBD, either alone or in combination with other immunomodulators.

PGE2 enhanced TNFα-mediated IL-8 induction in monocytic cell lines and PBMC.

BACKGROUND & PURPOSE: Recent studies suggested a role of prostaglandin E2 (PGE2) in the expression of the chemokine IL-8 by monocytes. The function of EP4 receptor for TNFα-induced IL-8 expression was studied in monocytic cell lines. EXPERIMENTAL APPROACH: IL-8 mRNA and protein induction as well as IL-8 promoter activity and transcription factor activation were assessed in monocytic cell lines, primary blood mononuclear cells (PBMC) and transgenic HEK293 cells expressing the EP4 receptor. KEY RESULTS: In monocytic cell lines THP-1, MonoMac and U937 PGE2 had only a marginal impact on IL-8 induction but strongly enhanced TNFα-induced IL-8 mRNA and protein synthesis. Similarly, in PBMC IL-8 mRNA induction was larger by simultaneous stimulation with TNFα and PGE2 than by either stimulus alone. The EP4 receptor subtype was the most abundant EP receptor in all three cell lines and in PBMC. Stimulation of THP-1 cells with an EP4 specific agonist enhanced TNFα-induced IL-8 mRNA and protein formation to the same extent as PGE2. In HEK293 cells expressing EP4, but not in wild type HEK293 cells lacking EP4, PGE2 enhanced TNFα-induced IL-8 protein and mRNA synthesis. In THP-1 cells, the enhancement of TNFα-mediated IL-8 mRNA induction by PGE2 was mimicked by a PKA-activator. Furthermore in these cells PGE2 induced expression of transcription factor C/EBPß, enhanced NF-κB activation by TNFα and inhibited TNFα-mediated AP-1 activation. PGE2 and TNFα synergistically activated transcription factor CREB, induced C/EBPß expression and enhanced the activity of an IL-8 promoter fragment containing -223 bp upstream of the transcription start site. CONCLUSIONS AND IMPLICATIONS: These findings suggest that a combined stimulation of TNFα and PGE2/EP4 signal chains in monocytic cells leads to maximal IL-8 promoter activity, as well as IL-8 mRNA and protein induction, by activating the PKA/CREB/C/EBPß as well as NF-κB signal chains.

The regulatory role of C1q on Helicobacter pylori-induced inflammatory cytokines secretion in THP-1 cells.

C1q, as a LAIR-1 ligand, maintains monocytes quiescence and possess immunosuppressive properties. To understand the roles and molecular mechanisms, C1q mediated inflammation cytokines and several pivotal proteins in THP-1 cells after H. pylori infection were detected. The results showed that the expression of IL-8, IL-10, LAIR-1, phosphorylated/total JNK, phosphorylated/total p38-MAPK, phosphorylated/total AKT and phosphorylated/total NF-κB were up-regulated significantly in THP-1 cells after H. pylori infection. There was significant upregulation in IL-10 concentration, phosphorylated/total p38-MAPK and phosphorylated/total AKT, and downregulation in phosphorylated/total JNK in non-H. pylori infected THP-1 cells pretreated with C1q. C1q was also able to increase IL-8 and IL-10 production, and reduce LAIR-1 and phosphorylated/total p38-MAPK expression in pretreatment-C1q THP-1 cells after H. pylori infection. These results together indicated that H. pylori might induce IL-8 and IL-10 production through JNK, p38-MAPK, PI3K/AKT and NF-κB signaling pathway. C1q manipulate LAIR-1 to regulation IL-8 and IL-10 secretion in THP-1 cells after H. pylori infection through the p38-MAPK signaling pathway. This information is helpful to further understand the role and mechanisms of C1q on inflammation cytokines secretion in monocytes after H. pylori infection.

An MD2-derived peptide promotes LPS aggregation, facilitates its internalization in THP-1 cells, and inhibits LPS-induced pro-inflammatory responses.

MD2, a 160-residue accessory glycoprotein, is responsible for the recognition and binding of Gram-negative bacterial membrane component, lipopolysaccharide (LPS). Internalization of pathogen inside the mononuclear phagocytes has also been attributed to MD2 which leads to the clearance of pathogens from the host. However, not much is known about the segments in MD2 that are responsible for LPS interaction or internalization of pathogen inside the defense cells. A 16-residue stretch (MD54) from MD2 protein has been identified that possesses a short heptad repeat sequence and four cationic residues enabling it to participate in both hydrophobic and electrostatic interactions with LPS. An MD54 analog of the same size was also designed in which a leucine residue at a heptadic position was replaced with an alanine residue. MD54 but not its analog, MMD54 induced aggregation of LPS and aided in its internalization within THP-1 monocytes. Furthermore, MD54 inhibited LPS-induced nuclear translocation of NF-κB in PMA-treated THP-1 and TLR4/MD2/CD14-transfected HEK-293T cells and the production of pro-inflammatory cytokines. In addition, in in vivo experiments, MD54 showed marked protection and survival of mice against LPS-induced inflammation and death. Overall, we have identified a short peptide with heptad repeat sequence from MD2 that can cause aggregation of LPS and abet in its internalization within THP-1 cells, resulting in attenuation of LPS-induced pro-inflammatory responses in vitro and in vivo.

Matrine Suppresses Reactive Oxygen Species (ROS)-Mediated MKKs/p38-Induced Inflammation in Oxidized Low-Density Lipoprotein (ox-LDL)-Stimulated Macrophages.

BACKGROUND The objective of this study was to study the anti-inflammatory effect and possibly involved molecular mechanisms of matrine on oxidized low-density lipoprotein (ox-LDL)-exposed macrophages. MATERIAL AND METHODS Cultured human macrophages (THP-1 cell line) were exposed to ox-LDL at final concentrations of 0, 25, 50, and 100 µg/mL. Several cells were then treated with matrine at serial diluted concentrations. 2,7-Dichlorodi-hydrofluorescein diacetate (DCFH-DA) staining was used to evaluate reactive oxygen species (ROS) production; a colorimetric method was used to determine the cellular antioxidant capacity; production of pro-inflammatory cytokines interleukin (IL)18 and tumor necrosis factor (TNF)alpha were determined by enzyme-linked immunosorbent assay (ELISA); and immunoblot assay was used to assess the relative protein phosphorylation and expression. RESULTS ox-LDL exposure significantly elevated intracellular ROS level and supernatant IL18 and TNFalpha concentrations, but impaired total antioxidant capacity (TAC) of macrophages. The relative phosphorylations of MAPK kinase kinases (MKK)6, MKK3, and p38 mitogen-activated protein kinases (MAPK) were increased by ox-LDL exposure. The expression levels of IL18 and TNFalpha were also increased in ox-LDL-treated macrophages. The matrine treatment reduced intracellular ROS level and supernatant IL18 and TNFalpha concentrations and increased TAC in a concentration- dependent manner. The relative phosphorylations of MKK6, MKK3, and p38 MAPK were reduced after matrine administration. Moreover, the expression levels of IL18 and TNFalpha were also decreased by matrine treatment, in a concentration-dependent manner. CONCLUSIONS ox-LDL increases inflammatory response in macrophages by activating the ROS-mediated MKKs/p38 MAPK-induced inflammatory signaling pathway. Matrine suppresses ox-LDL-induced inflammatory by inhibiting the MKKs/p38 MAPK signaling pathway.

Curdepsidones B⁻G, Six Depsidones with Anti-Inflammatory Activities from the Marine-Derived Fungus Curvularia sp. IFB-Z10.

Six new depsidones, curdepsidones B-G (1-6), were obtained from the marine-derived fungus Curvularia sp. IFB-Z10. Their planar structures were determined by comprehensive analysis of HRESIMS and 1D/2D-NMR data. The absolute configuration of curdepsidones B-C (1-2) were established by synergistic use of DFT/NMR (density functional theory/nuclear magnetic resonance) and TDDFT/ECD (time-dependent density functional theory/electronic circular dichroism) calculations. Partial isolated compounds were tested for their anti-inflammatory activities in Propionibacterium acnes-induced THP-1 cells. Curdepsidone C (2) displayed significant anti-inflammatory properties with an IC50 value of 7.47 ± 0.35 µM, and reduced the P. acnes-induced phosphorylation levels of JNK and ERK in a dose-dependent mechanism. The possible anti-inflammatory mechanism of 2 was also investigated by molecular docking.

Hirsutanol A Attenuates Lipopolysaccharide-Mediated Matrix Metalloproteinase 9 Expression and Cytokines Production and Improves Endotoxemia-Induced Acute Sickness Behavior and Acute Lung Injury.

Activated human monocytes/macrophages, which increase the levels of matrix metalloproteinases (MMPs) and pro-inflammatory cytokines, are the essential mechanisms for the progression of sepsis. In the present study, we determined the functions and mechanisms of hirsutanolA (HA), which is isolated from the red alga-derived marine fungus Chondrostereum sp. NTOU4196, on the production of pro-inflammatory mediators produced from lipopolysaccharide (LPS)-treated THP-1 cells. Our results showed that HA suppressed LPS-triggered MMP-9-mediated gelatinolysis and expression of protein and mRNA in a concentration-dependent manner without effects on TIMP-1 activity. Also, HA significantly attenuated the levels of TNF-α, IL-6, and IL-1ß from LPS-treated THP-1 cells. Moreover, HA significantly inhibited LPS-mediated STAT3 (Tyr705) phosphorylation, IκBα degradation and ERK1/2 activation in THP-1 cells. In an LPS-induced endotoxemia mouse model, studies indicated that HA pretreatment improved endotoxemia-induced acute sickness behavior, including acute motor deficits and anxiety-like behavior. HA also attenuated LPS-induced phospho-STAT3 and pro-MMP-9 activity in the hippocampus. Notably, HA reduced pathologic lung injury features, including interstitial tissue edema, infiltration of inflammatory cells and alveolar collapse. Likewise, HA suppressed the induction of phospho-STAT3 and pro-MMP-9 in lung tissues. In conclusion, our results provide pharmacological evidence that HA could be a useful agent for treating inflammatory diseases, including sepsis.

SAR-mediated Similarity Assessment of the Property Profile for New, Silicon-Based AChE/BChE Inhibitors.

A set of 25 novel, silicon-based carbamate derivatives as potential acetyl- and butyrylcholinesterase (AChE/BChE) inhibitors was synthesized and characterized by their in vitro inhibition profiles and the selectivity indexes (SIs). The prepared compounds were also tested for their inhibition potential on photosynthetic electron transport (PET) in spinach (Spinacia oleracea L.) chloroplasts. In fact, some of the newly prepared molecules revealed comparable or even better inhibitory activities compared to the marketed drugs (rivastigmine or galanthamine) and commercially applied pesticide Diuron®, respectively. Generally, most compounds exhibited better inhibition potency towards AChE; however, a wider activity span was observed for BChE. Notably, benzyl N-[(1S)-2-[(tert-butyldimethylsilyl)oxy]-1-[(2-hydroxyphenyl)carbamoyl]ethyl]-carbamate (2) and benzyl N-[(1S)-2-[(tert-butyldimethylsilyl)oxy]-1-[(3-hydroxyphenyl)carbamoyl]ethyl]-carbamate (3) were characterized by fairly high selective indexes. Specifically, compound 2 was prescribed with the lowest IC50 value that corresponds quite well with galanthamine inhibition activity, while the inhibitory profiles of molecules 3 and benzyl-N-[(1S)-2-[(tert-butyldimethylsilyl)oxy]-1-[(4-hydroxyphenyl)carbamoyl]ethyl]carbamate (4) are in line with rivastigmine activity. Moreover, a structure-activity relationship (SAR)-driven similarity evaluation of the physicochemical properties for the carbamates examined appeared to have foreseen the activity cliffs using a similarity-activity landscape index for BChE inhibitory response values. The 'indirect' ligand-based and 'direct' protein-mediated in silico approaches were applied to specify electronic/steric/lipophilic factors that are potentially valid for quantitative (Q)SAR modeling of the carbamate analogues. The stochastic model validation was used to generate an 'average' 3D-QSAR pharmacophore pattern. Finally, the target-oriented molecular docking was employed to (re)arrange the spatial distribution of the ligand property space for BChE and photosystem II (PSII).

Magnesium sulfate inhibits binding of lipopolysaccharide to THP-1 cells by reducing expression of cluster of differentiation 14.

We investigated effects of magnesium sulfate (MgSO4) on modulating lipopolysaccharide (LPS)-macrophage binding and cluster of differentiation 14 (CD14) expression. Flow cytometry data revealed that the mean levels of LPS-macrophage binding and membrane-bound CD14 expression (mCD14) in differentiated THP-1 cells (a human monocytic cell line) treated with LPS plus MgSO4 (the LPS + M group) decreased by 28.2% and 25.3% compared with those THP-1 cells treated with LPS only (the LPS group) (P < 0.001 and P = 0.037), indicating that MgSO4 significantly inhibits LPS-macrophage binding and mCD14 expression. Notably, these effects of MgSO4 were counteracted by L-type calcium channel activation. Moreover, the mean level of soluble CD14 (sCD14; proteolytic cleavage product of CD14) in the LPS + M group was 25.6% higher than in the LPS group (P < 0.001), indicating that MgSO4 significantly enhances CD14 proteolytic cleavage. Of note, serine protease inhibition mitigated effects of MgSO4 on both decreasing mCD14 and increasing sCD14. In conclusion, MgSO4 inhibits LPS-macrophage binding through reducing CD14 expression. The mechanisms may involve antagonizing L-type calcium channels and activating serine proteases.

Candidate Polyurethanes Based on Castor Oil (Ricinus communis), with Polycaprolactone Diol and Chitosan Additions, for Use in Biomedical Applications.

Polyurethanes are widely used in the development of medical devices due to their biocompatibility, degradability, non-toxicity and chemical versatility. Polyurethanes were obtained from polyols derived from castor oil, and isophorone diisocyanate, with the incorporation of polycaprolactone-diol (15% w/w) and chitosan (3% w/w). The objective of this research was to evaluate the effect of the type of polyol and the incorporation of polycaprolactone-diol and chitosan on the mechanical and biological properties of the polyurethanes to identify the optimal ones for applications such as wound dressings or tissue engineering. Polyurethanes were characterized by stress-strain, contact angle by sessile drop method, thermogravimetric analysis, differential scanning calorimetry, water uptake and in vitro degradation by enzymatic processes. In vitro biological properties were evaluated by a 24 h cytotoxicity test using the colorimetric assay MTT and the LIVE/DEAD kit with cell line L-929 (mouse embryonic fibroblasts). In vitro evaluation of the possible inflammatory effect of polyurethane-based materials was evaluated by means of the expression of anti-inflammatory and proinflammatory cytokines expressed in a cellular model such as THP-1 cells by means of the MILLIPLEX® MAP kit. The modification of polyols derived from castor oil increases the mechanical properties of interest for a wide range of applications. The polyurethanes evaluated did not generate a cytotoxic effect on the evaluated cell line. The assessed polyurethanes are suggested as possible candidate biomaterials for wound dressings due to their improved mechanical properties and biocompatibility.

TNF-α-Induced NOD2 and RIP2 Contribute to the Up-Regulation of Cytokines Induced by MDP in Monocytic THP-1 Cells.

Nucleotide-binding oligomerization domain containing 2 (NOD2)-induced signal transduction and cytokine production is regulated by a number of factors. However, the feedback effect of the pro-inflammatory TNF-α on NOD2-induced inflammation is not fully understood. In this study, we found unexpectedly that TNF-α up-regulated NOD2 ligand MDP-induced production of the CXC chemokines, including CXCL1, 2, and 8, and the pro-inflammatory cytokines, including IL-1ß, IL-6, and TNF-α, in a dose-dependent manner at both mRNA and protein levels in monocytic THP-1 cells. Though TNF-α induced the up-regulation of ubiquitin-editing enzyme A20, an important negative regulator for Toll-like receptor- and NOD2-induced inflammatory responses, the over-expression of A20 by gene transfer did not reversed MDP-induced production of cytokines, suggested that A20 did not regulate the functions of NOD2 in THP-1 cells. Meanwhile, we found that TNF-α up-regulated NOD2 and its down-stream adaptor protein RIP2 at both mRNA and protein levels. MDP induced the activation of ERK, JNK, p38 and NF-κB, and TNF-α pre-treatment augmented this activation. The results from pharmacological inhibition assay showed that cytokine production was dependent on MAPK signaling. In addition, we found that the pre-treatment of THP-1 cells with MDP down-regulated the mRNA levels of cytokine induced by MDP re-treatment. MDP pre-treatment up-regulated NOD2, but down-regulated RIP2, and down-regulated NOD2 signal transduction induced by MDP re-stimulation. Taking together, these results suggested that TNF-α is a positive regulator for NOD2 functions via up-regulation of NOD2 and its signal adaptor RIP2, and TNF-α-induced A20 does not regulate MDP-induced inflammatory responses in THP-1 cells. J. Cell. Biochem. 119: 5072-5081, 2018. © 2017 Wiley Periodicals, Inc.

A20 restores phorbol ester-induced differentiation of THP-1 cells in the absence of nuclear factor-κB activation.

A20, also referred to as tumor necrosis factor alpha (TNFα)-induced protein 3 (TNFAIP3), is an ubiquitin-editing enzyme whose expression is enhanced by NF-κB activation, and plays an important role in silencing NF-κB activity. Another well-known role for A20 is to protect cells from TNFα-induced apoptosis. Depletion of NF-κB in differentiating U937 monocytic leukemia cells is known to cause apoptotic cell death; however, much remains to be explored about the molecules that are expressed in an NF-κB-dependent manner and which support monocyte-macrophage differentiation. Using the monocytic cell line THP-1, and peripheral blood monocytes, we show here a sustained increase in A20 expression during monocyte-macrophage differentiation, which coincided with high NF-κB-dependent transcriptional activity. Depletion of NF-κB by stable expression of a super-repressor form of IκBα in THP-1 cells caused remarkable cell death during phorbol 12-myristate 13-acetate (PMA)-induced differentiation. A20 expression in these cells did not alter this NF-κB suppression, but was sufficient to protect the cells and restore the cell surface expression of a differentiation marker (CD11b) and phagocytic activity. Mutational analyses revealed that this A20 activity requires the carboxy-terminal zinc-finger domain, but not its deubiquitinase activity. Based on these findings, we conclude that A20, when ectopically expressed, can support both survival and differentiation of THP-1 cells in the absence of sustained NF-κB activity.

Characterizing the Effects of Glutathione as an Immunoadjuvant in the Treatment of Tuberculosis.

Mycobacterium tuberculosis is the etiological agent that is responsible for causing tuberculosis (TB), which continues to affect millions of people worldwide, and the rate of resistance of M. tuberculosis to antibiotics is ever increasing. We tested the synergistic effects of N-acetyl cysteine (NAC; the precursor molecule for the synthesis of glutathione [GSH]) and individual first-line antibiotics typically given for the treatment of TB, such as isoniazid (INH), rifampin (RIF), ethambutol (EMB), and pyrazinamide (PZA), to improve the ability of macrophages to control intracellular M. tuberculosis infection. GSH, a pleiotropic antioxidant molecule, has previously been shown to display both antimycobacterial and immune-enhancing effects. Our results indicate that there was not only an increase in beneficial immunomodulatory effects but also a greater reduction in the intracellular viability of M. tuberculosis when macrophages were treated with the combination of antibiotics (INH, RIF, EMB, or PZA) and NAC.