Dataset Information

Secretome of P2X7 receptor - Macrophage activation and polarization modify P2X7 receptor secretome influencing the inflammatory process

ABSTRACT: In order to determine P2X7R secretome we analyzed the proteins present in cell-free supernatants from wild-type (P2rx7+/+) or P2rx7-/- bone marrow-derived macrophages (BMDMs) polarized either to M1 or M2 and subsequently treated with ATP. BMDMs were primed with LPS (M1) or IL-4 (M2) for 4 hours and the proteins secreted during this step were extensively washed with PBS before ATP was added in fresh buffer. The complex mixture of proteins obtained in the macrophages supernatants after ATP stimulation were fractionated using one dimension gel electrophoresis and 10 bands were selected for LC-MS/MS analysis based in their presence in higher intensity in P2rx7+/+ supernatant compared with P2rx7-/- supernatant.

INSTRUMENT(S): Thermo Finnigan instrument model

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Bone Marrow, Macrophage

SUBMITTER: Carlos de Torre Minguela

LAB HEAD: Carlos de Torre Minguela

PROVIDER: PXD001981 | Pride | 2016-03-11

REPOSITORIES: Pride

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Publications

Macrophage activation and polarization modify P2X7 receptor secretome influencing the inflammatory process.

de Torre-Minguela Carlos C Barberà-Cremades Maria M Gómez Ana I AI Martín-Sánchez Fátima F Pelegrín Pablo P

Scientific reports 20160303

The activation of P2X7 receptor (P2X7R) on M1 polarized macrophages induces the assembly of the NLRP3 inflammasome leading to the release of pro-inflammatory cytokines and the establishment of the inflammatory response. However, P2X7R signaling to the NLRP3 inflammasome is uncoupled on M2 macrophages without changes on receptor activation. In this study, we analyzed P2X7R secretome in wild-type and P2X7R-deficient macrophages polarized either to M1 or M2 and proved that proteins released after P ...[more]

PMID: 26935289

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Project description:To recruit phagocytes, apoptotic cells characteristically release ATP, which functions as a “danger” signal. Here, we found that the culture supernatant of apoptotic cells activated the macrophages to express anti-inflammatory genes such as NR4A and Thbs1. A high level of AMP accumulated in the apoptotic cell supernatant in a Pannexin1-dependent manner. A nucleotidase inhibitor and A2a adenosine receptor antagonist inhibited the apoptotic supernatant-induced gene expression, suggesting AMP was metabolized to adenosine by an ecto-5’-nucleotidase expressed on macrophages, to activate the macrophage A2a adenosine receptor. Intraperitoneal injection of zymosan into AdoR A2a- or Panx1-deficient mice produced high, sustained levels of inflammatory mediators in the peritoneal lavage. These results indicated that AMP from apoptotic cells suppresses inflammation as a “calm down” signal. If apoptotic cells produce “danger” or “anti-danger” signal(s), we rationalized that such signals would activate gene expression in macrophages. To investigate this possibility, we examined the effect of the culture supernatant from apoptotic cells on macrophage gene expression by using microarrays. For mouse BMDMs, bone marrow cells from female C57BL/6J mice at 8 weeks of age were cultured for more than 7 days with DMEM containing 10% FCS supplemented with mouse M-CSF. We used adherent cells as BMDMs in the study. W3 cells, mouse T cell line expressing Fas, were treated with human Fas ligand at 37°C for 30 min to induce apoptosis. The cells were then washed and re-suspended at a concentration of 1 × 107 cells/ml with RPMI containing 1% FCS, and further incubated for 60 min at 37°C. Following Fas ligand treatment, more than 90% of the W3 cells were Annexin V positive, and only small percentage were positive for both Annexin V and propidium iodide (PI). The culture supernatant was collected from apoptotic W3 cells. Next, BMDMs were incubated with medium (BMDMs-Medium) or apoptotic W3 cell supernatant (BMDMs-Apoptotic cell supernatant) for 1 h. Total RNA was extracted from the cells and hybridized on Affymetrix microarrays.

Dataset Information

Secretome of P2X7 receptor - Macrophage activation and polarization modify P2X7 receptor secretome influencing the inflammatory process

Publications

Macrophage activation and polarization modify P2X7 receptor secretome influencing the inflammatory process.

Similar Datasets

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