Chemoattractant Signaling between Tumor Cells and Macrophages Regulates Cancer Cell Migration, Metastasis and Neovascularization

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-1α 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

GARCINIA COWA ROXB. ETHANOL EXTRACT INHIBITS INFLAMMATION IN LPS-INDUCED RAW 264.7 MACROPHAGES

Objective: The purpose of this study was to examine the effect of Garcinia cowa Roxb. Ethanol (EGC) extract in LPS-induced Raw 264.7 macrophages by observing the release of Tumor Necrosis Factor (TNF) and Interleukin-6 (IL-6). Methods: Using the MTT method, a cell viability assay was performed to observe the cytotoxic effect on Raw 264.7 macrophages. For 24 h, Raw 264.7 macrophages were incubated with various EGC concentrations (100, 50, 10, 1 and 0.1 µg/ml). The medium was taken out after 48 h of incubation, and 100 µl of MTT 0.5 mg/ml was then added. 100 µl DMSO was used to dissolve the crystals and absorbance was measured using a microplate reader. To investigate the activity of EGC to LPS-induced Raw 264.7 macrophages, the ELISA method was used. Supernatant was obtained after treating Raw 264.7 macrophages with complete medium, EGC samples, and LPS (10 g/ml) for 24 h. IL-6 and TNF-α levels were assessed using supernatants with ELISA kit. Results: Cytotoxic effect of EGC to Raw 264.7 macrophages occurred at a concentration of 100 µg/ml with the cell viability value of 59.5%. At a concentration of 50 µg/ml, no cytotoxic effect occurred and the cell viability value was 105.5%. So, the higher concentration of EGC used for further investigation is 50 µg/ml. It was shown that the production of IL 6 was suppressed by EGC at a concentration of 12.5 µg/ml. The inhibition of TNF-α production was only seen at the concentration of 12.5, 25 and 50 µg/ml; there was an increase of TNF-α production. Conclusion: It can be concluded that EGC can be developed as a natural immunomodulator that can inhibit inflammation by suppressing IL-6 production to prevent immune system disorders

Platelet-Rich Fibrin Reduces IL-1β Release from Macrophages Undergoing Pyroptosis.

BACKGROUND Pyroptosis is a catabolic process relevant to periodontal disorders for which interleukin-1β (IL-1β) inflammation is central to the pathophysiology of the disease. Despite platelet-rich fibrin (PRF) anti-inflammatory properties and its application to support periodontal regeneration, the capacity of PRF to modulate pyroptosis, specifically the production and release of IL-1β, remains unknown. The question arises whether PRF could regulate IL-1β release from macrophages in vitro. METHODS To answer this question, RAW 264.7 macrophages and primary macrophages obtained from murine bone marrow were primed with PRF before being challenged by lipopolysaccharide (LPS). Cells were then analysed for the pyroptosis signalling components by gene expression analyses and IL-1β secretion at the protein level. The release of mitochondrial reactive oxygen species (ROS) was also detected. RESULTS PRF lowered the LPS-induced expression of IL-1β and NLRP3 inflammasome, caspase-11 and IL-18 in primary macrophages, and IL-1β and caspase-11 in RAW 264.7 cells. Additionally, PRF diminished the secretion of IL-1β at the protein level in LPS-induced RAW 264.7 cells. This was shown through immunoassays performed with the supernatant and further confirmed by analysing the lysates of permeabilised cells. Furthermore, PRF reduced the ROS release provoked by LPS in RAW 264.7 cells. Finally, to enhance IL-1β release from the LPS-primed macrophages, we introduced a second signal with adenosine triphosphate (ATP). In this setting, PRF significantly reduced IL-1β release in RAW 264.7 cells and a trend to diminish IL-1β release in primary macrophages. CONCLUSION These findings suggest that PRF can reduce IL-1β release and, at least in part, inhibit pyroptosis-related factors in LPS-challenged macrophages

High-resolution quantitative proteome analysis reveals substantial differences between phagosomes of RAW 264.7 and bone marrow-derived macrophages

Macrophages are important immune cells operating at the forefront of innate immunity by taking up foreign particles and microbes through phagocytosis. The RAW 264.7 cell line is commonly used for experiments in the macrophage and phagocytosis field. However, little is known how its functions compare to primary macrophages. Here, we have performed an in‐depth proteomics characterization of phagosomes from RAW 264.7 and bone marrow derived macrophages by quantifying more than 2500 phagosomal proteins. Our data indicate that there are significant differences for a large number of proteins including important receptors such as mannose receptor 1 and Siglec‐1. Moreover, bone marrow derived macrophages phagosomes mature considerably faster by fusion with endosomes and the lysosome which we validated using fluorogenic phagocytic assays. We provide a valuable resource for researcher in the field and recommend careful use of the RAW 264.7 cell line when studying phagosome functions. All MS data have been deposited in the ProteomeXchange with identifier PXD001293 (http://proteomecentral.proteomexchange.org/dataset/PXD001293)

Inhibitory effects of Gynura procumbens ethanolic extract on nitric oxide production and inducible nitric oxide synthase (iNOS) protein expression in macrophages

Nitric oxide (NO) overproduction by inducible nitric oxide synthase (iNOS) may be associated with acute and chronic inflammations. Macrophages as important cells in the innate immune system are able to be stimulated and can lead to iNOS activation and excessive NO production. Gynura procumbens is a medicinal plant traditionally used in treating various ailments including inflammation but the mechanism of anti-inflammatory activity of this plant is still elusive. This study was carried out to investigate the anti-inflammatory therapeutic effects of Gynura procumbens ethanolic extract on NO production and iNOS protein expression in RAW 264.7 macrophages stimulated with lipopolysaccharide (LPS). Cell viability of RAW 264.7 macrophages treated with Gynura procumbens ethanolic extract was determined by MTT assay. NO production was determined by Griess assay following Gynura procumbens ethanolic extract treatment alone or in combination with LPS stimulation. Protein expression of iNOS was determined by western blot. RAW 264.7 macrophages viability of more than 90% was observed after 24 h treatment with Gynura procumbens ethanolic extract concentration range of 3.9 μg/mL to 500 μg/mL. Significant inhibition of NO production level has been identified in LPS-stimulated RAW 264.7 cells pre-treated with 250 μg/mL Gynura procumbens ethanolic extract (p<0.05) while all selected concentrations of Gynura procumbens ethanolic extract showed no significant alteration of NO production in the absence of LPS stimulation. Pre-treatment of 250 μg/mL Gynura procumbens ethanolic extract also demonstrated significant suppression of iNOS protein expression in LPS-stimulated RAW 264.7 cells (p<0.05). In conclusion, this study demonstrates that Gynura procumbens ethanolic extract exhibits anti-inflammatory potential through inhibition of NO production and iNOS protein expression in LPS-stimulated macrophages, suggesting that this plant could be further researched for its beneficial use in inflammatory disorders

Elucidating the Role of HDAC8 in Anthrax Lethal Toxin-Induced Pyroptosis and Cytokine Gene Silencing in Macrophages

Anthrax is a lethal infectious disease caused by the bacterium Bacillus anthracis. B. anthracis secretes the virulence factor anthrax lethal toxin (LeTx), which causes rapid cell death known as pyroptosis and immune suppression in macrophages. Strikingly, RAW 264.7 macrophages pre-exposed to sub-lethal doses of LeTx become refractory to subsequent high cytolytic doses. The phenomenon is termed toxin-induced resistance (TIR). TIR is in part linked to the down-regulation of three mitochondrial death genes, BCL2/adenovirus E1B 19 kDa-interacting protein 3 (BNIP3), BNIP3-like (BNIP3L), and metastatic lymph node 64 (MLN64) protein, as well as the up-regulation of a gene-silencing epigenetic regulator, histone deacetylase (HDAC) 8. Interestingly, I found that inhibiting HDAC8 with the HDAC8-specific inhibitor PCI-34051 in RAW 264.7 TIR cells sensitized them to LeTx-induced pyroptosis. Furthermore, resistance to LeTx-induced pyroptosis is likely mediated by HDAC8-dependent H3K27Ac deacetylation in the regulatory regions of the mitochondrial death genes, BNIP3 and MLN64. Although, sub-lethal doses of LeTx induced resistance to pyroptosis in RAW 264.7 macrophages, LeTx still caused immune suppression. Here, I found that RAW 264.7 LeTx-treated macrophages and RAW 264.7 macrophages over-expressing HDAC8 failed to produce pro-interleukin (IL)-1β, a pro-inflammatory cytokine, in response to the Gram-negative bacterial cell wall component lipopolysaccharide (LPS), whereas inhibiting HDAC8 with PCI-34051 restored IL-1β and tumor necrosis factor (TNF)-α production in response to LPS. Furthermore, chromatin immunoprecipitation (ChIP)- quantitative real-time polymerase chain reaction (qPCR) analysis revealed that inhibiting HDAC8 in the presence of LeTx increased H3K27Ac association with the genomic regions of IL-1β in response to LPS. Collectively, these results suggest that HDAC8 plays a key role in resistance to LeTx-induced pyroptosis and LeTx-induced immunosuppression, through targeting the H3K27Ac-associated regions of BNIP3, MLN64 and IL-1β

Synergistic effect of phosphodiesterase 4 inhibitor and serum on migration of endotoxin-stimulated macrophages.

Macrophage migration is an essential step in host defense against infection and wound healing. Elevation of cAMP by inhibiting phosphodiesterase 4 (PDE4), enzymes that specifically degrade cAMP, is known to suppress various inflammatory responses in activated macrophages, but the role of PDE4 in macrophage migration is poorly understood. Here we show that the migration of Raw 264.7 macrophages stimulated with LPS was markedly and dose-dependently induced by the PDE4 inhibitor rolipram as assessed by scratch wound healing assay. Additionally, this response required the involvement of serum in the culture medium as serum starvation abrogated the effect. Further analysis revealed that rolipram and serum exhibited synergistic effect on the migration, and the influence of serum was independent of PDE4 mRNA expression in LPS-stimulated macrophages. Moreover, the enhanced migration by rolipram was mediated by activating cAMP/exchange proteins directly activated by cAMP (Epac) signaling, presumably via interaction with LPS/TLR4 signaling with the participation of unknown serum components. These results suggest that PDE4 inhibitors, together with serum components, may serve as positive regulators of macrophage recruitment for more efficient pathogen clearance and wound repair

Increased S-nitrosylation and proteasomal degradation of caspase-3 during infection contribute to the persistence of adherent invasive escherichia coli (AIEC) in immune cells

Adherent invasive Escherichia coli (AIEC) have been implicated as a causative agent of Crohn's disease (CD) due to their isolation from the intestines of CD sufferers and their ability to persist in macrophages inducing granulomas. The rapid intracellular multiplication of AIEC sets it apart from other enteric pathogens such as Salmonella Typhimurium which after limited replication induce programmed cell death (PCD). Understanding the response of infected cells to the increased AIEC bacterial load and associated metabolic stress may offer insights into AIEC pathogenesis and its association with CD. Here we show that AIEC persistence within macrophages and dendritic cells is facilitated by increased proteasomal degradation of caspase-3. In addition S-nitrosylation of pro- and active forms of caspase-3, which can inhibit the enzymes activity, is increased in AIEC infected macrophages. This S-nitrosylated caspase-3 was seen to accumulate upon inhibition of the proteasome indicating an additional role for S-nitrosylation in inducing caspase-3 degradation in a manner independent of ubiquitination. In addition to the autophagic genetic defects that are linked to CD, this delay in apoptosis mediated in AIEC infected cells through increased degradation of caspase-3, may be an essential factor in its prolonged persistence in CD patients