Expression data from BNL CL.2 liver cells when cultured with iron, RAW 264.7 macrophages or both
ABSTRACT: We found that co-culturing BNL CL.2 liver cells with RAW 264.7 macrophages increased IRP binding in the first. To further investigate this modulation we investigated the gene expression profile in BNL CL.2 cells cultured alone, with iron, with RAW 264.7 macrophages or in the presence of both iron and macrophages. This novel reconstituted liver cell-macrophage communication pathway with the present gene expression data provides a platform for addressing how macrophages participate in the iron homeostasis of liver cells and, ultimately, in systemic iron homeostasis. We used microarrays to determine the gene expression modulation in BNL CL.2 cells in response to 24h culture with 100 micromolar ferric ammonium citrate (FAC), co-culture with RAW 264.7 macrophages or both
Project description:We found that co-culturing BNL CL.2 liver cells with RAW 264.7 macrophages increased IRP binding in the first. To further investigate this modulation we investigated the gene expression profile in BNL CL.2 cells cultured alone, with iron, with RAW 264.7 macrophages or in the presence of both iron and macrophages. This novel reconstituted liver cell-macrophage communication pathway with the present gene expression data provides a platform for addressing how macrophages participate in the iron homeostasis of liver cells and, ultimately, in systemic iron homeostasis. We used microarrays to determine the gene expression modulation in BNL CL.2 cells in response to 24h culture with 100 micromolar ferric ammonium citrate (FAC), co-culture with RAW 264.7 macrophages or both
Project description:While nanoparticles (NPs) are increasingly used in a variety of consumer products and medical applications, some of these materials have potential health concerns. Macrophages are the primary responders to particles that initiate oxidative stress and inflammatory reactions. Here, we utilized six flame-synthesized, engineered iron oxide NPs with various physicochemical properties (e.g. Fe oxidation state and crystal size) to study their interactions with RAW 264.7 macrophages, their iron solubilities, and their abilities to produce hydroxyl radical in an acellular assay. Both iron solubility and hydroxyl radical production varied between NPs depending on crystalline diameter and surface area of the particles, but not on iron oxidation state. Macrophage treatment with the iron oxide NPs showed a dose-dependent increase of heme oxygenase 1 (HO-1) and NAD(P)H:quinone oxidoreductase (NQO-1). The nuclear factor (NF)-erythroid-derived 2 (E2)-related factor 2 (Nrf2) modulates the transcriptional activity of antioxidant response element (ARE)-driven genes, such as HO-1 and NQO-1. Here, we show that the iron oxide NPs activate Nrf2, leading to its increased nuclear accumulation and enhanced Nrf2 DNA-binding activity in NP-treated RAW 264.7 macrophages. Iron solubility and acellular hydroxyl radical generation depend on the physical properties of the NPs, especially crystalline diameter; however, these properties are weakly linked to the activation of cellular signaling of Nrf2 and the expression of oxidative stress markers. Overall, our work shows for the first time that iron oxide nanoparticles induce cellular marker genes of oxidative stress and that this effect is transcriptionally mediated through the Nrf2-ARE signaling pathway in macrophages.
Project description:The critical function of ferroportin (Fpn) in maintaining iron homeostasis requires complex and multilevel control of its expression. Besides iron-dependent cellular and systemic control of Fpn expression, other metals also seem to be involved in regulating the <i>Fpn</i> gene. Here, we found that copper loading significantly enhanced <i>Fpn</i> transcription in an Nrf2-dependent manner in primary bone-marrow-derived macrophages (BMDMs). However, prolonged copper loading resulted in decreased Fpn protein abundance. Moreover, CuCl<sub>2</sub> treatment induced Fpn expression in RAW 264.7 macrophages at both the mRNA and protein level. These data suggest that cell-type-specific regulations have an impact on Fpn protein stability after copper loading. Transcriptional suppression of Fpn after lipopolysaccharide (LPS) treatment contributes to increased iron storage inside macrophages and may result in anemia of inflammation. Here, we observed that in both primary BMDMs and RAW 264.7 macrophages, LPS treatment significantly decreased Fpn mRNA levels, but concomitant CuCl<sub>2</sub> stimulation counteracted the transcriptional suppression of Fpn and restored its expression to the control level. Overall, we show that copper loading significantly enhances <i>Fpn</i> transcription in macrophages, while Fpn protein abundance in response to CuCl<sub>2</sub> treatment, depending on macrophage type and factors specific to the macrophage population, can influence Fpn regulation in response to copper loading.
Project description:Immunoresponsive gene 1 (IRG1) is one of the highest induced genes in macrophages under pro-inflammatory conditions and its function has been recently described: it codes for immune-responsive gene 1 protein/cis-aconitic acid decarboxylase (IRG1/CAD), an enzyme catalyzing the production of itaconic acid from cis-aconitic acid, a tricarboxylic acid (TCA) cycle intermediate. Itaconic acid possesses specific antimicrobial properties inhibiting isocitrate lyase, the first enzyme of the glyoxylate shunt, an anaplerotic pathway that bypasses the TCA cycle and enables bacteria to survive on limited carbon conditions. To elucidate the mechanisms underlying itaconic acid production through IRG1 induction in macrophages, we examined the transcriptional regulation of IRG1. Using a combination of literature information, transcription factor prediction models and genome-wide expression arrays, we inferred the regulatory network of IRG1 in mouse and human macrophages. 3 unstimulated (Control) and 3 LPS-stimulated RAW 264.7 macrophages
Project description:Tumor-associated macrophages are known to influence cancer progression by modulation of immune function, angiogenesis, and cell metastasis, however, little is known about the chemokine signaling networks that regulate this process. Utilizing CT26 colon cancer cells and RAW 264.7 macrophages as a model cellular system, we demonstrate that treatment of CT26 cells with RAW 264.7 conditioned medium induces cell migration, invasion and metastasis. Inflammatory gene microarray analysis indicated CT26-stimulated RAW 264.7 macrophages upregulate SDF-1alpha and VEGF, and that these cytokines contribute to CT26 migration in vitro. RAW 264.7 macrophages also showed a robust chemotactic response towards CT26-derived chemokines. In particular, microarray analysis and functional testing revealed CSF-1 as the major chemoattractant for RAW 264.7 macrophages. Interestingly, in the chick CAM model of cancer progression, RAW 264.7 macrophages localized specifically to the tumor periphery where they were found to increase CT26 tumor growth, microvascular density, vascular disruption, and lung metastasis, suggesting these cells home to actively invading areas of the tumor, but not the hypoxic core of the tumor mass. In support of these findings, hypoxic conditions down regulated CSF-1 production in several tumor cell lines and decreased RAW 264.7 macrophage migration in vitro. Together our findings suggest a model where normoxic tumor cells release CSF-1 to recruit macrophages to the tumor periphery where they secrete motility and angiogenic factors that facilitate tumor cell invasion and metastasis.
Project description:'Zhique' (Citrus wilsonii Tanaka) is a traditional Chinese medicine. Its fruits have been used to treat inflammation-related symptoms, such as cough and sputum, though the underlying mechanism remains poorly understood. The aim of this study was to investigate the anti-inflammatory properties of 'Zhique' pulp extract (ZQE) in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages and primary mouse bone marrow-derived dendritic cells (BMDCs). The flavonoid profiles of the ZQE were determined by high performance liquid chromatography. The anti-inflammatory activity was evaluated in LPS-induced inflammatory RAW 264.7 macrophages and BMDCs through enzyme-linked immunosorbent assay, quantitative real-time polymerase chain reaction, and Western blot assays. Naringin was a predominant flavonoid occurring in ZQE, followed by eriocitrin, hesperidin, neohesperidin, rhoifolin, naringenin, and poncirin. ZQE exhibited a very low cytotoxicity in LPS-stimulated RAW 264.7 macrophages. Meanwhile, ZQE significantly inhibited the production of prostaglandins E2 and secretion of cyclooxygenase-2 protein in LPS-stimulated RAW 264.7 macrophages, and markedly suppressed the mRNA expression of inflammatory mediators, such as cyclooxygenase-2, tumor necrosis factor alpha, interleukin-1 beta (IL-1?), and IL-6 in LPS-induced RAW 264.7 macrophages and/or primary BMDCs. The ZQE inhibited the inflammatory responses in RAW 264.7 macrophages and BMDCs triggered by LPS. The results suggested that 'Zhique' has a high potential as a novel therapeutic agent to treat chronic inflammatory diseases.
Project description:Macrophages are activated by IFN-gamma, a proinflammatory and proatherogenic cytokine that mediates its downstream effects primarily through STAT1. IFN-gamma signaling induces phosphorylation of two STAT1 residues: Tyr(701) (Y701), which facilitates dimerization, nuclear translocation, and DNA binding; and Ser(727) (S727), which enables maximal STAT1 transcription activity. Immunosuppressive molecules such as adenosine in the cellular microenvironment can reduce macrophage inflammatory and atherogenic functions through receptor-mediated signaling pathways. We hypothesized that adenosine achieves these protective effects by interrupting IFN-gamma signaling in activated macrophages. This investigation demonstrates that adding adenosine to IFN-gamma-stimulated murine RAW 264.7 and human THP-1 macrophages results in unique modulation of STAT1 serine and tyrosine phosphorylation events. We show that adenosine inhibits IFN-gamma-induced STAT1 S727 phosphorylation by >30% and phosphoserine-mediated transcriptional activity by 58% but has no effect on phosphorylation of Y701 or receptor-associated JAK tyrosine kinases. Inhibition of the adenosine A(3) receptor with a subtype-specific antagonist (MRS 1191 in RAW 264.7 cells and MRS 1220 in THP-1 cells) reverses this adenosine suppressive effect on STAT1 phosphoserine status by 25-50%. Further, RAW 264.7 A(3) receptor stimulation with Cl-IB-MECA reduces IFN-gamma-induced STAT1 transcriptional activity by 45% and STAT1-dependent gene expression by up to 80%. These data suggest that A(3) receptor signaling is key to adenosine-mediated STAT1 modulation and anti-inflammatory action in IFN-gamma-activated mouse and human macrophages. Because STAT1 plays a key role in IFN-gamma-induced inflammation and foam cell transformation, a better understanding of the mechanisms underlying STAT1 deactivation by adenosine may improve preventative and therapeutic approaches to vascular disease.
Project description:In this study, we isolated sargachromenol (SC) from <i>Sargassum horneri</i> and evaluated its anti-inflammatory effect in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. SC did not show cytotoxicity at all concentrations and effectively increased the cell viability by reducing the nitric oxide (NO) and intracellular reactive oxygen species (ROS) production in LPS-stimulated RAW 264.7 macrophages. In addition, SC decreased the mRNA expression levels of inflammatory cytokines (IL-1β, IL-6, and TNF-α) and inflammatory mediators (iNOS and COX-2). Moreover, SC suppressed the activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) and mitogen-activated protein kinase (MAPK) signaling, whereas activated the nuclear factor erythroid 2-related factor 2/heme oxygenase-1 (Nrf2/HO-1) signaling in LPS-stimulated RAW 264.7 macrophages. Interestingly, the anti-inflammatory effect of SC was abolished by the inhibition of HO-1 in LPS-stimulated RAW 264.7 macrophages. According to the results, this study suggests that the antioxidant capacity of SC leads to its anti-inflammatory effect and it potentially may be utilized in the nutraceutical and pharmaceutical sectors.
Project description:We inflicted TBI to chemokine-deficient mouse lines in order to establish involvement of various signalling pathways that may be addressed therapeutically. Interacting chemokine pathways in brain regulate distinct inflammatory cells. Activated microglia are separate from invading phagocytes and dendritic cells. Findings show potential targets to interfere with specific inflammatory responses after brain injury. TBI was carried out in Ccl3-/- and Ccr2-/- mice, total RNA prepared from injured cerebral neocortex after three days. RNA samples were from uninjured Ccl3-/- and Ccr2-/- mice as reference for hybridization on Affymetrix microarrays.