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Phenotypic changes in bone marrow-derived murine macrophages cultured on PEG-based hydrogels activated or not by lipopolysaccharide.

ABSTRACT: Macrophages are phenotypically diverse cells performing a number of functions involved in immunity, inflammation, wound healing, tissue homeostasis and the foreign body reaction. In the latter, the type of biomaterial and the surrounding environment likely have an impact on macrophage phenotype and, subsequently, the severity of the reaction. The objectives for this study were to characterize the phenotype of bone marrow-derived murine macrophages in response to poly(ethylene glycol) (PEG)-based hydrogels, a promising class of materials for cell delivery. Gene expression was used as a measure of phenotype and characterized by IL-1?, TNF-?, iNOS, IL-12?, arginase, VEGF-A, and IL-10. Macrophages were cultured on PEG hydrogels, PEG hydrogels with RGD tethers, and medical grade silicone rubber, a well-characterized biomaterial, up to 96 h in the absence and presence of lipopolysaccharide (LPS) to simulate an inflammatory environment. Macrophage interrogation led to immediate up-regulation (10×) of IL-1? and TNF-? within 4h, followed by an increase in IL-10/IL-12? and a subsequent concomitant decrease in the pro-inflammatory genes by 96 h, suggesting a shift from classically activated to a regulatory phenotype. LPS stimulation led to a stronger early up-regulation of pro-inflammatory genes (e.g. 20-30× for IL-1? and TNF-?), followed by upregulation (4-6×) of arginase, suggesting a shift from an elevated classically activated to a wound healing phenotype. Material type played a significant role in regulating pro-inflammatory genes, which was most pronounced with PEG alone. Overall, our findings indicate that macrophages undergo similar phenotypic changes for the materials tested, but the magnitudes of these responses are highly material dependent.

SUBMITTER: Lynn AD

PROVIDER: S-EPMC2967672 | biostudies-literature | 2011 Jan

REPOSITORIES: biostudies-literature

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Phenotypic changes in bone marrow-derived murine macrophages cultured on PEG-based hydrogels activated or not by lipopolysaccharide.

Lynn Aaron D AD Bryant Stephanie J SJ

Acta biomaterialia 20100730 1

Macrophages are phenotypically diverse cells performing a number of functions involved in immunity, inflammation, wound healing, tissue homeostasis and the foreign body reaction. In the latter, the type of biomaterial and the surrounding environment likely have an impact on macrophage phenotype and, subsequently, the severity of the reaction. The objectives for this study were to characterize the phenotype of bone marrow-derived murine macrophages in response to poly(ethylene glycol) (PEG)-based ...[more]

PMID: 20674808

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Project description:BackgroundPeriodontal tissue regeneration is the ultimate goal of periodontitis treatment. Exosomes are nanoscale vesicles secreted by cells that participate in and regulate the physiological activities between cells. However, the relationship between inflammatory macrophage-derived exosomes and osteoblast differentiation in periodontitis has not been thoroughly reported. Here, we attempt to explore the role of inflammatory macrophage-derived exosomes in crosstalk with osteoblasts.MethodsPorphyromonas gingivalis lipopolysaccharide was used to stimulate macrophages and inflate their inflammatory cellular state. Exosomes were extracted from inflammatory macrophages using supercentrifugation, and their characteristics were detected by transmission electron microscopy, particle size analysis, and Western blotting. Exosome uptake bybone marrow mesenchymal stem cells (BMSCs) was observed by fluorescence microscopy. The effects of exosomes on the BMSC inflammatory response and on osteogenic differentiation were detected by quantitative polymerase chain reaction and Western blot analysis. Alkaline phosphatase activity was tested for verification.ResultsWe successfully extracted and identified inflammatory macrophage-derived exosomes and observed that BMSCs successfully took up exosomes. Inflammatory macrophage-derived exosomes upregulated the expression levels of the inflammatory factors interleukin-6 and tumour necrosis factor-alpha in BMSCs and mediated inflammatory stimulation. Additionally, they inhibited the transcription levels of the osteogenic genes alkaline phosphatase, type I collagen, and Runt-related transcription factor 2 as well as the alkaline phosphatase activity, while the use of the exosome inhibitor GW4869 attenuated this effect.ConclusionOur study shows that macrophages in periodontitis can mediate inflammatory stimulation and inhibit the osteogenic differentiation of bone marrow mesenchymal stem cells through the exosome pathway. Interference with exosome secretion is likely to be a promising method for bone tissue regeneration in inflammatory states.

Project description:Published data show that murine bone marrow-derived macrophages (BMDM) restrict growth of avirulent phase II, but not virulent phase I, Coxiella burnetii. Growth restriction of phase II bacteria is thought to result from potentiated recognition of pathogen-associated molecular patterns, which leads to production of inhibitory effector molecules. Past studies have used conditioned medium from L-929 murine fibroblasts as a source of macrophage-colony stimulating factor (M-CSF) to promote differentiation of bone marrow-derived myeloid precursors into macrophages. However, uncharacterized components of conditioned medium, such as variable amounts of type I interferons, can affect macrophage activation status and their permissiveness for infection. In the current study, we show that the C. burnetii Nine Mile phase II (NMII) strain grows robustly in primary macrophages from C57BL/6J mice when bone marrow cells are differentiated with recombinant murine M-CSF (rmM-CSF). Bacteria were readily internalized by BMDM, and replicated within degradative, LAMP1-positive vacuoles to achieve roughly 3 logs of growth over 6 days. Uninfected BMDM did not appreciably express CD38 or Egr2, markers of classically (M1) and alternatively (M2) activated macrophages, respectively, nor did infection change the lack of polarization. In accordance with an M0 phenotype, infected BMDM produced moderate amounts of TNF and nitric oxide. Similar NMII growth results were obtained using C57BL/6J myeloid progenitors immortalized with an estrogen-regulated Hoxb8 (ER-Hoxb8) oncogene. To demonstrate the utility of the ER-Hoxb8 system, myeloid progenitors from natural resistance-associated macrophage protein 1 (Nramp1) C57BL/6J knock-in mice were transduced with ER-Hoxb8, and macrophages were derived from immortalized progenitors using rmM-CSF and infected with NMII. No difference in growth was observed when compared to macrophages from wild type mice, indicating depletion of metal ions by the Nramp1 transporter does not negatively impact NMII growth. Results with NMII were recapitulated in primary macrophages where C57BL/6J Nramp1+ BMDM efficiently killed Salmonella enterica serovar Typhimurium. M-CSF differentiated murine macrophages from bone marrow and conditional ER-Hoxb8 myeloid progenitors will be useful ex vivo models for studying Coxiella-macrophage interactions.

Dataset Information

Phenotypic changes in bone marrow-derived murine macrophages cultured on PEG-based hydrogels activated or not by lipopolysaccharide.

Publications

Phenotypic changes in bone marrow-derived murine macrophages cultured on PEG-based hydrogels activated or not by lipopolysaccharide.

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