Primary Murine Peritoneal Macrophages Research Articles

Cyasterone ameliorates sepsis-related acute lung injury via AKT (Ser473)/GSK3β (Ser9)/Nrf2 pathway

BackgroundAcute lung injury (ALI) is a severe disease that can lead to acute respiratory distress syndrome (ARDS), characterized by intractable hypoxemia, poor lung compliance, and respiratory failure, severely affecting patients' quality of life. The pathogenesis of ALI has not been fully elucidated yet, and sepsis is an important cause of ALI. Among the organ injuries caused by sepsis, the lungs are the earliest damaged ones. Radix cyathulae is reported to have analgesic, anti-inflammatory, and anti-aging effects. Cyasterone is extracted from Radix cyathulae. However, it is not known whether cyasterone has protective effects for ALI. This study aims to investigate the effect of cyasterone on sepsis-related ALI and its mechanism.MethodsWe used the cecal ligation peferation (CLP) method to establish a mouse sepsis model, and cyasterone was given intraperitoneally on days 1–3 to observe its preventive effect on sepsis-related acute lung injury. Primary murine peritoneal macrophages were used to investigate the molecular mechanism of cyasterone in vitro.ResultsCyasterone pretreatment inhibits pro-inflammatory cytokine production, NLRP3 inflammasome activation, and oxidative stress in vivo and in vitro. In addition, cyasterone attenuates sepsis-induced ALI by activating nuclear factor erythroid2-related factor (Nrf2), which may be associated with AKT(Ser473)/GSK3β(Ser9) pathway activation.ConclusionsCyasterone defends against sepsis-induced ALI by inhibiting inflammatory responses and oxidative stress, which depends heavily on the upregulation of the Nrf2 pathway through phosphorylation of AKT(Ser473)/GSK3β(Ser9). These results suggest cyasterone may be a valuable drug candidate for preventing sepsis-related ALI.

Extracellular CIRP Induces Macrophage Extracellular Trap Formation Via Gasdermin D Activation.

Extracellular cold-inducible RNA-binding protein (eCIRP) is a damage-associated molecular pattern promoting inflammation and tissue injury. During bacterial or viral infection, macrophages release DNA decorated with nuclear and cytoplasmic proteins known as macrophage extracellular traps (METs). Gasdermin D (GSDMD) is a pore-forming protein that has been involved in extracellular trap formation in neutrophils. We hypothesized that eCIRP induces MET formation by activating GSDMD. Human monocytic cell line THP-1 cells were differentiated with phorbol 12-myristate 13-acetate (PMA) and treated with recombinant murine (rm) CIRP. The MET formation was detected by three methods: time-lapse fluorescence microscopy (video imaging), colorimetry, and ELISA. Cleaved forms of GSDMD, and caspase-1 were detected by Western blotting. Treatment of THP-1 cells with rmCIRP increased MET formation as revealed by SYTOX Orange Staining assay in a time- and dose-dependent manner. METs formed by rmCIRP stimulation were further confirmed by extracellular DNA, citrullinated histone H3, and myeloperoxidase. Treatment of THP-1 cells with rmCIRP significantly increased the cleaved forms of caspase-1 and GSDMD compared to PBS-treated cells. Treatment of macrophages with caspase-1, and GSDMD inhibitors z-VAD-fmk, and disulfiram, separately, significantly decreased rmCIRP-induced MET formation. We also confirmed rmCIRP-induced MET formation using primary cells murine peritoneal macrophages. These data clearly show that eCIRP serves as a novel inducer of MET formation through the activation of GSDMD and caspase-1.

Interferon-gamma impairs phagocytosis of Escherichia coli by primary murine peritoneal macrophages stimulated with LPS and differentially modulates proinflammatory cytokine release

IntroductionInterferon-γ levels are increased upon viral infections and during inflamm-aging. Resistance to infections due to Escherichia coli (E. coli), a major cause of bacteriaemia and sepsis, is impaired in aged individuals, partly due to altered phagocytic capacity and cytokine release of immune cells. Here, we analyzed the effect of IFN-γ on phagocytosis of E. coli K1 and release of proinflammatory cytokines by macrophages in resting condition and upon stimulation with different bacterial Toll-like receptor (TLR) agonists. MethodsPrimary peritoneal macrophages from C57BL/6 mice were exposed to medium or stimulated with agonists of TLR4 (LPS), 1/2 (Pam3CSK4), and 9 (CpG-DNA) in the presence and absence of IFN-γ (100 U/ml) for 24 h. TNF-α, IL-6, and KC were measured in the cell culture supernatant by ELISA. Macrophages were exposed to viable E. coli K1. After 90 min, intracellular phagozytosed bacteria were quantified by quantitative plating. ResultsMacrophages treated with LPS 1 µg/ml in the presence of IFN-γ ingested more than 10-fold lower numbers of E. coli than macrophages treated with LPS alone. Phagocytosis of E. coli by macrophages in resting condition or upon stimulation with Pam3CSK4 or CpG was not significantly affected by IFN-γ. Cytokine release was differentially modulated by IFN-γ, with reduced KC release by TLR-stimulated macrophages in the presence of IFN-γ being the most striking effect. ConclusionsIn vitro, IFN-γ reduces the phagocytosis of E. coli by LPS-stimulated macrophages and differentially modulates cytokine release of macrophages activated by different bacterial TLR agonists. Elevated levels of IFN-γ might lead to reduced bacterial clearance and worse outcome of bacterial infections, e.g., in aged individuals and after viral infections and other inflammatory events.

Loganin alleviates sepsis-induced acute lung injury by regulating macrophage polarization and inhibiting NLRP3 inflammasome activation

Sepsis is a systemic inflammatory response syndrome resulted from severe infection. Excessive inflammation response plays an important role in sepsis-induced acute lung injury (ALI). Loganin is an iridoid glycoside isolated from Corni fructus and exerts an anti-inflammatory effect in multiple inflammatory diseases; however, the role of loganin in sepsis-induced ALI remains unknown. In the current study, the cecal ligation and puncture (CLP)-induced murine sepsis model was constructed to investigate the anti-inflammatory property of loganin in sepsis-induced ALI. Lipopolysaccharide (LPS)-treated Raw 264.7 cells and primary murine peritoneal macrophages were established to further explore underlying mechanism of loganin. Results showed that intragastrical administration of loganin significantly increased murine survival, reduced the alveolar structure damage and inflammatory cell infiltration. Loganin suppressed the release of the M1 macrophage-associated pro-inflammatory cytokines and induced the activation of M2-type anti-inflammatory cytokines. Besides, loganin dramatically inhibited NLRP3 inflammasome-mediated caspase-1 activation and subsequent IL-1β secretion. Further in vitro studies confirmed that loganin efficiently inhibited M1 macrophage polarization and NLRP3 inflammasome activation by blocking the extra-cellular signal-regulated kinase (ERK) and nuclear factor-kappa B (NF-κB) pathways. Taken together, the anti-inflammatory effect of loganin in sepsis-induced ALI was associated with the ERK and NF-κB pathway-mediated macrophage polarization and NLRP3 inflammasome activation. Our study offers a favorable mechanistic basis to support the therapeutic potential of loganin in anti-inflammatory diseases, such as sepsis-induced ALI.

Pro-Inflammatory Effects of Indoxyl Sulfate in Mice: Impairment of Intestinal Homeostasis and Immune Response.

The intestines are recognized as the main source of chronic inflammation in chronic kidney disease (CKD) and, among other cells, macrophages are involved in modulating this process as well as in the impaired immune response which also occurs in CKD patients. In this study, we evaluated the effect of Indoxyl Sulfate (IS), a protein bound uremic toxin poorly eliminated by hemodialysis, on inflammatory, oxidative stress and pro-apoptotic parameters, at the intestinal level in mice, on intestinal epithelial cells (IEC-6) and on primary murine peritoneal macrophages. C57BL/6J mice were treated with IS (800 mg/kg i.p.) for 3 or 6 h and histopathological analysis showed that IS induced intestinal inflammation and increased cyclooxygenase-2 (COX-2), nitrotyrosine and Bax expression in intestinal tissue. In IEC-6 cells, IS (125–1000 µM) increased tumor necrosis factor-α levels, COX-2 and inducible nitric oxide synthase expression and nitrotyrosine formation. Moreover, IS increased pro-oxidant, pro-inflammatory and pro-apoptotic parameters in peritoneal macrophages from IS-treated mice. Also, the serum concentration of IS and pro-inflammatory levels of cytokines resulted increased in IS-treated mice. Our results indicate that IS significantly contributes to affect intestinal homeostasis, immune response, and to induce a systemic pro-inflammatory state thus highlighting its potential role as therapeutic target in CKD patients.

JNK‑IN‑8 treatment alleviates lipopolysaccharide‑induced acute lung injury via suppression of inflammation and oxidative stress regulated by JNK/NF‑κB signaling.

JNK serves critical roles in numerous types of inflammation- and oxidative stress-induced disease, including acute lung injury (ALI). JNK-IN-8 is the first irreversible JNK inhibitor that has been described. However, whether JNK-IN-8 can prevent lipopolysaccharide (LPS)-induced ALI by inhibiting JNK activation and its downstream signaling is poorly understood. The objective of the present study was to investigate the specific therapeutic effects of JNK-IN-8 on LPS-induced ALI and the molecular mechanisms involved. JNK-IN-8 attenuated myeloperoxidase activity, malondialdehyde and superoxide dismutase content and the lung wet/dry ratio, and improved the survival rate following lethal injection of LPS. Additionally, JNK-IN-8 decreased bronchoalveolar lavage fluid protein levels, lactate dehydrogenase activity, neutrophil infiltration and the number of macrophages (as demonstrated by flow cytometry), as well as the production of TNF-α, IL-6 and IL-1β (as evaluated via ELISA). In addition, reverse transcription-quantitative PCR and ELISA showed that JNK-IN-8 attenuated LPS-induced inflammatory cytokine production and oxidative stress in primary murine peritoneal macrophages and RAW264.7 cells in vitro. Furthermore, the present study demonstrated that the JNK/NF-κB signaling pathway was involved in the therapeutic effect of JNK-IN-8 against LPS-induced injury both in vivo and in vitro. In conclusion, these findings indicated that JNK-IN-8 had a therapeutic effect on LPS-induced ALI in mice. The mechanism may be associated with inhibition of the JNK/NF-κB signaling pathway. JNK-IN-8 may be a potential therapeutic agent for the treatment of ALI.

The Thickness of Surface Grafting Layer on Bio-materials directly Mediates the Immuno-reacitivity of Macrophages in vitro.

Introducing the surface grafting layers to regulate bio-compatibility and bio-function is an important step in the tissue engineering field. However, whether the thickness variation of the introduced biomimetic layer impacts the behavior of the adhered immune effector cells is yet to be dissected fully. In this study, we used a surface-induced atom transfer radical polymerization (SI-ATRP) method to synthetize and graft poly-phenoxyethyl methacrylate (PHEMA) brushes having different lengths on the glass substrates. Primary murine peritoneal macrophages were collected and cultured on the PHEMA brushes and we investigated the influence of polymer brushes having different lengths on macrophages phenotype and function. Our results demonstrated that the thicker brushes (200 nm and 450 nm) are superior to the thinner layers (50 nm) for macrophages survival, proliferation, cell elongation and migration. Moreover, the thicker brushes are more beneficial for macrophage’s activities and functions, presented by the increased production of M1-associated cytokines IL-6 and MCP-1, the elevated cell phagocytosis and the activation molecule F4/80 expression, and the reduced macrophages apoptosis in thicker brushes-sustained macrophages. Our data suggests that the thickness of the substrate grafting layer directly impacts macrophages recruitment and pro-inflammatory function, which is important in determining the intrinsic immuno-compatibilities of the surface modified-biomaterials and mediates material-host interactions in vivo.

The therapeutic effect of verapamil in lipopolysaccharide-induced acute lung injury

The objective of this study was to investigate the exact therapeutic effects of Verapamil on lipopolysaccharide (LPS)-induced acute lung injury (ALI) and the molecular mechanism involved, through using LPS-induced animal models as well as LPS-stimulated mouse primary peritoneal macrophages models. Our results demonstrated that Verapamil reduced LPS-induced pathological damage of the lung tissue, infiltration of inflammatory cells and the production of IL-1β, TNF-α, and MCP-1 in the serum. The MPO activity, MDA content, lung wet/dry ratio and LDH activity were also attenuated by Verapamil. In addition, Verapamil attenuated LPS-induced inflammatory cytokine production and oxidative stress in primary murine peritoneal macrophages in vitro. Moreover, we confirmed that NF-κB/NLRP3 pathway was involved in the therapeutic effect of Verapamil against LPS-induced injury in vivo and in vitro. In conclusion, these findings indicate that Verapamil has a therapeutic effect on LPS-induced ALI in mice. The mechanism may be related to the inhibition of NF-κB and NLRP3 signaling pathways. Verapamil may be a potential therapeutic agent for the treatment of ALI.

Anti-inflammatory and diuretic effects of the diterpene ent-dihydrotucumanoic acid.

Gymnosperma glutinosum (Spreng) Less (Asteraceae) is a shrub used in traditional medicine for the treatment of inflammatory and renal diseases. The ent-dihydrotucumanoic acid (DTA) is a diterpene obtained from G. glutinosum. This study evaluated the antioxidant, genotoxic, and diuretic properties of DTA, as well as its in vitro and in vivo anti-inflammatory actions. The antioxidant actions of DTA were assessed with the 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), ferric reducing antioxidant power (FRAP), and 2,2'-diphenyl-1-picrylhydrazyl (DPPH) assays, the genotoxic action was assessed with the comet assay, and the diuretic effects of DTA were assessed using metabolic cages. The anti-inflammatory actions were evaluated using primary murine peritoneal macrophages stimulated with LPS and the λ-carrageenan-induced hind paw edema test. DTA lacked antioxidant (IC50 > 25,000 μg/mL) activity in the ABTS, FRAP, and DPPH assays. DTA at 500-1,000 μg/mL showed moderate genotoxicity. In LPS-stimulated macrophages, DTA showed IC50 values of 74.85 μg/mL (TNF-α) and 58.12 μg/mL (NO), whereas the maximum inhibition of IL-6 (24%) and IL-1β (36%) was recorded at 200 μg/mL. DTA induced in vivo anti-inflammatory effects with ED50 = 124.3 mg/kg. The in vitro anti-inflammatory activity of DTA seems to be associated with the decrease in the release of TNF-α and NO. DTA promoted the excretion of urine (ED50 = 86.9 mg/kg), Na+ (ED50 = 66.7 mg/kg), and K+ (ED50 = 8.6 mg/kg). The coadministration of DTA with L-NAME decreased the urinary excretion shown by DTA alone. Therefore, the diuretic activity is probably associated with the participation of nitric oxide synthase. In conclusion, DTA exerted anti-inflammatory and diuretic effects, but lacked antioxidant effects.

Assessment of Survival and Replication of Brucella spp. in Murine Peritoneal Macrophages.

Brucella spp. are bacteria that naturally infect a variety of domesticated and wild animals leading to abortions and infertility, and these bacteria are also capable of causing debilitating human infections, which often result from human exposure to infected animals and animal products. The brucellae are intracellular pathogens that reside in host cells, including macrophages and dendritic cells, and it is paramount for the pathogenesis of Brucella that the bacteria are able to survive and replicate in these host cells. The methods outlined in this chapter can be employed to study the interactions between Brucella strains and primary murine peritoneal macrophages.

Role of Toll-like receptor 4/oxidant-coupled activation in regulating the biosynthesis of omega-3 polyunsaturated fatty acid derivative resolvin D1 in primary murine peritoneal macrophage

We have previously shown that reactive oxygen species (ROS) as prooxidants can activate Toll-like receptor 4 (TLR4) with the potential to initiate, propagate and maintain “sterile” inflammation of innate immunity, which plays a mediatory role in a host of human disease states. We now present new evidence that ROS can also activate TLR4 to counter the inflammatory phenotype by increasing the production of resolvin D1 (RvD1), which is a specialized anti-inflammatory and pro-resolving lipid mediator. We used primary murine peritoneal macrophages (pM) derived from both TLR4-WT and TLR4-KO mice as a cellular model. We used potassium peroxychromate (PPC) as a direct in vitro source of exogenous ROS. PPC treatment increased intracellular ROS levels, which decreased intracellular total antioxidant capacity, thus suggesting an enhanced cellular oxidative stress. PPC and LPS-EK (a TLR4-specific agonist) increased pro-inflammatory TNFα production with noeffect on IL-10, an anti-inflammatory cytokine. Treatment with the prooxidant increased the expression of 12 lipoxygenase (12-LOX) and 5-lipoxygenase (5-LOX) only in pM derived from TLR4 WT but not in pM from TLR4-KO mice. 5-LOX and 12-LOX are the key enzymes in the RvD1 biosynthetic pathway. In addition, PPC increased the expression of RvD1 receptor, a member of G-protein-coupled receptor only in pM from TLR4-WT mice. Our data support the involvement of TLR4-mediated oxidant-induced pro-inflammatory phenotypes that are in opposition to the production of anti-inflammatory/pro-resolution phenotypes in macrophages. Now, we show that through TLR4 activation, exogenous oxidants can play a role both in producing proinflammatory phenotypes at the same time that it enhances resolution of inflammation to maintain a state of cellular homeostasis and prevent tissue damage/disease.

Lipopolysaccharide (LPS)-mediated priming of toll-like receptor 4 enhances oxidant-induced prostaglandin E2 biosynthesis in primary murine macrophages

Agonists and pseudo-agonists for toll-like receptor 4 (TLR4) are common in our environment. Thus, human exposure to these agents may result in “priming or sensitization” of TLR4. A body of evidence suggests that LPS-mediated sensitization of TLR4 can increase the magnitude of responses to exogenous agents in multiple tissues. We have previously shown that reactive oxygen and nitrogen species (RONS) stimulate TLR4. There is no evidence that LPS-primed TLR4 can influence the magnitude of responses to oxidants from either endogenous or exogenous sources. In the present study, we directly tested the hypothesis that LPS-primed TLR4 will sensitize primary murine peritoneal macrophages (pM) to oxidant-mediated prostaglandin E2 (PGE2) production. We used potassium peroxychromate (PPC) and potassium peroxynitrite (PPN) as direct in vitro sources of exogenous RONS. Our results showed that a direct treatment with PPC or PPN alone as sources of exogenous oxidants had a limited effect on PGE2 biosynthesis. In contrast, pM sensitized by prior incubation with LPS-EK, a TLR4-specific agonist, followed by oxidant stimulation exhibited increased transcriptional and translational expression of cyclooxygenase-2 (COX-2) with enhanced PGE2 biosynthesis/production only in pM derived from TLR4-WT mice but not in TLR4-KO mice. Thus, we have shown a critical role for LPS-primed TLR4 in oxidant-induced inflammatory phenotypes that have the potential to initiate, propagate and maintain many human diseases.

Calcitonin gene-related peptide exerts anti-inflammatory property through regulating murine macrophages polarization in vitro

Acute lung injury (ALI) is a condition resulting from direct or indirect lung injury associated with high mortality and morbidity. The phenotype of macrophages in lung contributes to the pathological progress of ALI. Calcitonin gene-related peptide (CGRP) is one of the most abundant neuropeptides in lung, and attenuates lipopolysaccharide (LPS)-induced ALI in rats. However, the exact effect of CGRP on the activation of macrophages remains unknown. Here we investigate the effect of CGRP on the macrophages activation and inflammation in murine macrophages in vitro. We found that LPS increased the expression of CGRP in a LPS-induced ALI murine model and LPS-stimulated murine macrophages. Although CGRP didn’t alter the expression of tumor necrosis factor-α (a marker of pro-inflammatory phenotype of macrophages, M1 macrophages) or Arginase 1 (Arg1, a marker of M2 macrophages) in non-differentiated macrophages, CGRP significantly reduced the NLRP3 and pro-IL-1β mRNA expression induced by LPS, as well as NLRP3 protein and IL-1β secretion induced by LPS+ATP in macrophages in vitro. On the other hand, CGRP dramatically enhanced the Arg1 expression and activity induced by IL-4 in the time- and dose-dependent manners. CGRP also promoted the expression of markers of M2 macrophages (IL-10, Fizz1 and Mrc1) induced by IL-4 in murine macrophages. These effects of CGRP were also observed in primary murine peritoneal macrophages. In addition, we found that CGRP regulated macrophages polarization partially through calmodulin, PKC and PKA pathways. Specifically, CGRP could inhibit the degradation of I-κB induced by LPS, and enhance the phosphorylation of STAT6 induced by IL-4 in macrophages. In conclusion, our results indicate that CGRP regulates macrophage polarization and inhibits inflammation in murine macrophages.

Abstract LB-295: A novel regulatory role of HDAC6 in the functional inflammatory phenotype of glia cells

Abstract Background: Histone Deacetylases (HDACs) have been demonstrated to modify a variety of non-histone proteins involved in oncogenic- and immune-related pathways. Recent studies in macrophages and tumor cells have implicated HDAC6 in the regulation of the immunomodulatory cytokines IL10 and IL6, and the immunosuppressive proteins PD-L1 and PD-L2. Additionally, specific inhibitors for this HDAC have shown promising results when used alone or in combination with other anticancer agents. Despite this newly characterized role of HDAC6 in immunobiology, its role in the regulation of immune-related pathways in the brain parenchymal environment is still unclear. Glia cells play a key role in a number of physiological processes in the brain, including the productions of inflammatory mediators and antigen presentation. Here, we report the first evidence of the functional effects of selective HDAC6inh in the modulation of the cytokine homeostasis and immune-related pathways in antigen presenting cells in the brain. Methods: Thioglycolate elicited peritoneal macrophages (PEM), primary murine cortical astrocytes, macrophage cell lines RAW264.7 and microglial cell line BV.2 were treated with the HDAC6 inhibitors Nexturastat and Tubastatin A, and the pan-HDACinh LBH589. Cytokine production was measured by qRT-PCR and ELISA. Viability, apoptosis, cell cycle, and HDAC activity was evaluated in parallel for all tested conditions. Results: In our comparative studies, the HDAC6 inhibition significantly lowered levels of cytokines IL6, IL10, IL1β, TNFα and IL12p70 levels in macrophages, and primary murine PEMs, indicating that both, anti- and pro-inflammatory cytokines are affected by this particular HDAC. Similar results were found in the expression of the pro-inflammatory cytokines IL1β, TNFα and IL12p70b in parallel studies using BV.2 microglia cells. In contrast, we observed an increase in the expression of IL6 and only slight variations in IL10 expression in BV.2, suggesting that HDAC6 might be controlling preferentially pro-inflammatory cytokines in glia cells. Additional control studies using the non-specific pan-HDACinh LBH589 showed similar outcomes in all analyzed cytokines in macrophages and glia cells. To further investigate the functional consequences of HDAC6inh in microglia cells, we evaluated the polarization phenotype of primary astrocytes and BV.2 cells under HDAC6inh treatment. While macrophages treated with Nexturastat and Tubastatin A were equally distributed between M1 and M2, we observed an important transition from M1 to M2 in brain cells, indicating a reduction in the pro-inflammatory phenotype of these cells when treated with HDAC6inh. Conclusion: Taken collectively, our studies demonstrate that HDAC6 plays a major role in the modulation of the functional phenotype and production of pro-inflammatory mediators in brain glia cells. These findings open the possibility for further evaluation of selective HDAC6inh as adjuvants in antitumor brain therapies. Citation Format: Jennifer Kim, Jayakumar R. Nair, Melissa M. Hadley, Alan Kozikowski, Robert Miller, Alejandro Villagra. A novel regulatory role of HDAC6 in the functional inflammatory phenotype of glia cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-295. doi:10.1158/1538-7445.AM2017-LB-295

Effect of bovine dialyzable leukocyte extract on induction of cell differentiation and death in K562 human chronic myelogenous leukemia cells.

Differentiation induction therapy is an attractive approach in leukemia treatment due to the fact that in blast crisis stage, leukemic cells lose their differentiation capacity. Therefore, it has been proposed as a therapeutic strategy to induce terminal differentiation of leukemic blast cells into a specific lineage, leading to prevention of high proliferation rates. The aim of the present study was to demonstrate the potential of cell differentiation and death induced by bovine dialyzable leukocyte extract (bDLE) in the K562 cell line. For this purpose K562 and MOLT-3 human leukemic cell lines and primary human monocytes and murine peritoneal macrophages were exposed to bDLE, phorbol myristate acetate (PMA) and dimethyl sulfoxide for 96 h, and the viability, proliferation and cell cycle were evaluated. To determine the lineage that led to cell differentiation, Romanowsky staining was performed to observe the morphological changes following the treatments, and the expression of the surface markers cluster of differentiation (CD)14+, CD68+, CD163+ and CD42a+, as well as the phagocytic activity, and the production of nitric oxide (NO) (assessed by colorimetric assay), cytokines [interleukin (IL)-1β, IL-6, IL-8 and tumor necrosis factor-α] and chemokines [chemokine (C-C motif) ligand (CCL)2, CCL5 and chemokine (C-X-C motif) ligand 8] in cell supernatants was assessed by flow cytometry. The results of the present study reveal that high doses of bDLE increase the cell death in K562 and MOLT-3 lines, without affecting the viability of human monocytes and murine peritoneal macrophages. Furthermore, low doses of bDLE induce differentiation in K562 cells towards a monocyte/macrophage lineage with an M2 phenotype, and induced moderately upregulated expression of CD42+, a megakaryocytic marker. Cell cycle arrest in the S and G2/M phases was observed in bDLE-treated K562 cells, which demonstrated similar phagocytic activity, NO levels and cytokine and chemokine production to that of PMA-treated cells. The present study demonstrates that bDLE exhibits an antileukemia effect, suggesting that it may be an effective candidate for leukemia treatment.

Type I interferon licenses enhanced innate recognition and transcriptional responses to Franciscella tularensis live vaccine strain.

The innate inflammatory response to Francisella tularensis (Ft) in macrophages is significantly governed by the expression of type I interferon (IFN). Previously, the proteolytic processing and maturation of pro-IL-1β protein was shown to depend upon type I IFN expression. We show in this report that paracrine type I IFN can profoundly enhance innate recognition and TLR-dependent transcriptional responses to Ft infection upstream of its role in inflammasome regulation in both primary human monocyte-derived macrophages and primary murine peritoneal macrophages but not murine bone marrow-derived macrophages. This type I IFN-enhanced response is synergistic with TLR2 transcriptional responses, partially TLR2-independent, but strictly MyD88-dependent.

Globular adiponectin inhibits ethanol-induced reactive oxygen species production through modulation of NADPH oxidase in macrophages: involvement of liver kinase B1/AMP-activated protein kinase pathway.

Adiponectin, an adipokine predominantly secreted from adipocytes, has been shown to play protective roles against chronic alcohol consumption. Although excessive reactive oxygen species (ROS) production in macrophages is considered one of the critical events for ethanol-induced damage in various target tissues, the effect of adiponectin on ethanol-induced ROS production is not clearly understood. In the present study, we investigated the effect of globular adiponectin (gAcrp) on ethanol-induced ROS production and the potential mechanisms underlying these effects of gAcrp in macrophages. Here we demonstrated that gAcrp prevented ethanol-induced ROS production in both RAW 264.7 macrophages and primary murine peritoneal macrophages. Globular adiponectin also inhibited ethanol-induced activation of NADPH oxidase. In addition, gAcrp suppressed ethanol-induced increase in the expression of NADPH oxidase subunits, including Nox2 and p22(phox), via modulation of nuclear factor-κB pathway. Furthermore, pretreatment with compound C, a selective inhibitor of AMPK, or knockdown of AMPK by small interfering RNA restored suppression of ethanol-induced ROS production and Nox2 expression by gAcrp. Finally, we found that gAcrp treatment induced phosphorylation of liver kinase B1 (LKB1), an upstream signaling molecule mediating AMPK activation. Knockdown of LKB1 restored gAcrp-suppressed Nox2 expression, suggesting that LKB1/AMPK pathway plays a critical role in the suppression of ethanol-induced ROS production and activation of NADPH oxidase by gAcrp. Taken together, these results demonstrate that globular adiponectin prevents ethanol-induced ROS production, at least in part, via modulation of NADPH oxidase in macrophages. Further, LKB1/AMPK axis plays an important role in the suppression of ethanol-induced NADPH oxidase activation by gAcrp in macrophages.

The Neoflavonoid Latifolin Isolated from MeOH Extract ofDalbergia odoriferaAttenuates Inflammatory Responses by Inhibiting NF-κB ActivationviaNrf2-Mediated Heme Oxygenase-1 Expression

In Korea and China, the heartwood of Dalbergia odorifera T. Chen is an important traditional medicine used to treat blood disorders, ischemia, swelling, and epigastric pain. In this study, we investigated the inhibitory effects of latifolin, a major neoflavonoid component isolated from the MeOH extract of D. odorifera, on the inflammatory reaction of thioglycollate-elicited peritoneal macrophages exposed to lipopolysaccharide, with a particular focus on heme oxygenase-1 (HO-1) expression and nuclear factor-κB (NF-κB) signaling. Latifolin significantly inhibited the protein and mRNA expression of inducible nitric oxide synthase and COX-2, reduced NO, prostaglandins E2, tumor necrosis factor-α, and interleukin-1β production in primary murine peritoneal macrophages exposed to lipopolysaccharide. Latifolin also suppressed inhibitor κB-α levels, NF-κB nuclear translocation, and NF-κB DNA-binding activity. Furthermore, latifolin upregulated HO-1 expression via nuclear transcription factor-E2-related factor 2 (Nrf2) nuclear translocation. In addition, using inhibitor tin protoporphyrin IX (SnPP), an inhibitor of HO-1, it was verified that the inhibitory effects of latifolin on the proinflammatory mediators and NF-κB DNA-binding activity were associated with the HO-1 expression. These results suggested that the latifolin-mediated up-regulation of HO-1 expression played a critical role in anti-inflammatory effects in macrophages. This study therefore identified potent therapeutic effects of latifolin, which warrants further investigation as a potential treatment for inflammatory diseases.

Ethanol increases matrix metalloproteinase-12 expression via NADPH oxidase-dependent ROS production in macrophages

Matrix metalloproteinase-12 (MMP-12), an enzyme responsible for degradation of extracellular matrix, plays an important role in the progression of various diseases, including inflammation and fibrosis. Although most of those are pathogenic conditions induced by ethanol ingestion, the effect of ethanol on MMP-12 has not been explored. In the present study, we investigated the effect of ethanol on MMP-12 expression and its potential mechanisms in macrophages. Here, we demonstrated that ethanol treatment increased MMP-12 expression in primary murine peritoneal macrophages and RAW 264.7 macrophages at both mRNA and protein levels. Ethanol treatment also significantly increased the activity of nicotinamide adenine dinucleotide (NADPH) oxidase and the expression of NADPH oxidase-2 (Nox2). Pretreatment with an anti-oxidant (N-acetyl cysteine) or a selective inhibitor of NADPH oxidase (diphenyleneiodonium chloride (DPI)) prevented ethanol-induced MMP-12 expression. Furthermore, knockdown of Nox2 by small interfering RNA (siRNA) prevented ethanol-induced ROS production and MMP-12 expression in RAW 264.7 macrophages, indicating a critical role for Nox2 in ethanol-induced intracellular ROS production and MMP-12 expression in macrophages. We also showed that ethanol-induced Nox2 expression was suppressed by transient transfection with dominant negative IκB-α plasmid or pretreatment with Bay 11-7082, a selective inhibitor of NF-κB, in RAW 264.7 macrophages. In addition, ethanol-induced Nox2 expression was also attenuated by treatment with a selective inhibitor of p38 MAPK, suggesting involvement of p38 MAPK/NF-κB pathway in ethanol-induced Nox2 expression. Taken together, these results demonstrate that ethanol treatment elicited increase in MMP-12 expression via increase in ROS production derived from Nox2 in macrophages.

Spermine oxidase is a regulator of macrophage host response to Helicobacter pylori: enhancement of antimicrobial nitric oxide generation by depletion of spermine

The gastric pathogen Helicobacter pylori causes peptic ulcer disease and gastric cancer. We have reported that in H. pylori-activated macrophages, nitric oxide (NO) derived from inducible NO synthase (iNOS) can kill the bacterium, iNOS protein expression is dependent on uptake of its substrate L-arginine (L-Arg), the polyamine spermine can inhibit iNOS translation by inhibiting L-Arg uptake, and inhibition of polyamine synthesis enhances NO-mediated bacterial killing. Because spermine oxidase (SMO), which back-converts spermine to spermidine, is induced in macrophages by H. pylori, we determined its role in iNOS-dependent host defense. SMO shRNA knockdown in RAW 264.7 murine macrophages resulted in a marked decrease in H. pylori-stimulated iNOS protein, but not mRNA expression, and a 90% reduction in NO levels; NO production was also inhibited in primary murine peritoneal macrophages with SMO knockdown. There was an increase in spermine levels after H. pylori stimulation that rapidly decreased, while SMO knockdown caused a greater increase in spermine that was sustained. With SMO knockdown, L-Arg uptake and killing of H. pylori by macrophages was prevented. The overexpression of SMO by transfection of an expression plasmid prevented the H. pylori-stimulated increase in spermine levels, and led to increased L-Arg uptake, iNOS protein expression and NO production, and H. pylori killing. In two human monocytic cell lines, U937 and THP-1, overexpression of SMO caused a significant enhancement of NO production with H. pylori stimulation. By depleting spermine, SMO can abrogate the inhibitory effect of polyamines on innate immune responses to H. pylori by enhancing antimicrobial NO production.