The P2X7 Receptor is a Master Regulator of Microparticle and Mitochondria Exchange in Mouse Microglia

Abstract

Microparticles (MPs) are secreted by all cells, where they play a key role in intercellular communication, differentiation, inflammation, and cell energy transfer. P2X7 receptor (P2X7R) activation by extracellular ATP (eATP) causes a large MP release and affects their contents in a cell-specific fashion. We investigated MP release and functional impact in microglial cells from P2X7R-WT or P2X7R-KO mice, as well as mouse microglial cell lines characterized for high (N13-P2X7RHigh) or low (N13-P2X7RLow) P2X7R expression. P2X7R stimulation promoted release of a mixed MP population enriched with naked mitochondria. Released mitochondria were taken up and incorporated into the mitochondrial network of the recipient cells in a P2X7R-dependent fashion. NLRP3 and the P2X7R itself were also delivered to the recipient cells. Microparticle transfer increased the energy level of the recipient cells and conferred a pro-inflammatory phenotype. These data show that the P2X7R is a master regulator of intercellular organelle and MP trafficking in immune cells.

Keywords: P2X7 receptor; inflammation; microglia; microparticles; mitochondria.

Graphical Abstract

Figure 1.

N13 microglial cells release MPs in a P2X7R-dependent fashion. Five ×10⁵ N13-P2X7R^High (A) or N13-P2X7R^Low (C) cells were loaded with the cytoplasmic marker calcein/AM (1 μg/mL) in RPMI complete culture medium for 20 min, then rinsed, re-suspended in the same medium, and challenged with 0.4 m m eATP for 15 min to trigger MP (arrow) release [(B, eATP-stimulated N13-P2X7R^High; D, eATP-stimulated N13-P2X7R^Low)]. (E-H) Cells were loaded with CSFE (5 μm) and incubated with increasing concentrations of eATP (E) or BzATP (F) for 60 min. (G) cells were treated with 0.3 m m oxidized ATP (oxo) for 1 h at 37°C prior to stimulation with either eATP or BzATP. (H) Time course of MP release in the presence or absence of either 0.4 m m eATP or 0.2 m m BzATP. To measure MP fluorescence, cell supernatants were withdrawn, centrifuged at 10 000 × g (4°C) for 1 h to concentrate the MPs, and the pellet re-suspended in 500 μL of sucrose medium. Fluorescence was measured in a fluorimeter cuvette at the 488/520 nm wavelength pair. Data are means ± SEM of three to four experiments each performed in triplicate. ** = P < 0.01; *** = P < 0.001; ^**** = P < 0.0001 by Student t-test (E-G), or two-way ANOVA (H). Fluorescence images were acquired with a Nikon Eclipse TE 300 inverted fluorescence microscope equipped with a 40× objective and fluorescein filters. Bar = 10 μm.

Figure 2.

Released MPs contain mitochondria. N13-P2X7R^High (A, B) cells (10⁵) were seeded onto 13-mm-diameter round glass coverslip and stained with the mitochondrial selective dye MitoTracker Green FM (200 n m) for 15 min at 37°C. The coverslips were then placed on the heated (37°C) stage of a Nikon inverted microscope as described in the legend to Figure 1, and either left unchallenged (A) or stimulated with 0.4 m m eATP for 5 min (B). Yellow arrows indicate MitoTracker Green FM-stained MPs. In panels C-F, supernatants from resting (C, D) or 0.4 m m eATP-stimulated (E, F) N13-P2X7^High were stained with calcein/AM (C, E) or MitoTracker Red FM (D, F) and analyzed with a Zeiss LSM510 confocal microscope equipped with a 63× oil immersion plan-apochromat objective. (G) Calcein/AM- or MitoTracker Red FM-positive MPs released from resting or eATP-stimulated N13-P2X7^High released over a 60-min timespan were quantitated. (H) Microparticles released over a 24 h incubation period from eATP (0.4 m m)-challenged N13-P2X7R^High cells co-labeled MitoTracker Red FM (200 n m) and calcein/AM (1 μm), were isolated as described in Materials and Methods and analyzed by confocal microscopy with rodhamine (upper) or fluorescein (middle) filters, and merged (lower). Microparticles isolated from N13-P2X7R^High were analyzed by TEM without (I) or with (J) immunogold labeling with an antibody against the specific mitochondrial protein TOM20. Microparticles isolated from resting (K) or 0.4 m m eATP-stimulated (L-N) N13-P2X7R^High were stained with TMRM and analyzed by confocal microscopy. 2.5 μm FCCP (M), or 200 n m rotenone (N) were also added. Bars = 20 μm (A-F), 10 μm (H, K-N). Data are means ± SEM from three independent experiments each performed in triplicate. ^****  P < 0.0001 by unpaired t test.

Figure 3.

Microparticle-P2X7R^High compared to MP-P2X7R^Low express higher levels of NLRP3, P2X7R and mitochondrial markers. N13-P2X7R^High and N13-P2X7R^Low cells were plated in 75 cm2 culture flasks and stimulated with 0.4 m m eATP or 0.2 m m BzATP for 24 h. At the end of this incubation, MPs were isolated and processed for Western Blotting analysis (A, B) as described in Materials and Methods. Carbonic anhydrase was used as a loading control. Densitometry (C-H) was performed with ImageJ software. mRNA was isolated from control or eATP-stimulated (0.4 m m) MPs (P2X7R, I), (NLRP3, J), and from MPs released during a 24-h incubation. CTRL: control resting cells. (K) Oxygen consumption by MP-P2X7R^High in the absence or presence of FCCP or oligomycin. Control (CTRL): O₂ consumption in the absence of MPs. (L) Intracellular ATP (iATP) content of MP-P2X7R^High and MP-P2X7R^Low. ATP was measured as described in Materials and Methods. Data are means ± SEM from three to four independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001; ^****  P < 0.0001 by unpaired t test.

Figure 4.

Released mitochondria are taken up by target N13 microglial cells in a P2X7R-dependent fashion. (A) Unlabeled N13-P2X7R^High or N13-P2X7R^Low were let adhere to 24-well dishes, rinsed, and further incubated in complete RPMI medium for 24 h with MitoTracker Green FM-labeled MP-P2X7R^High or MP-P2X7R^Low previously isolated from MitoTracker Green FM-labeled source N13 cells (see Materials and Methods). At the end of this incubation, the monolayers were rinsed to remove extracellular MPs, fresh medium was added, and the cells analyzed by confocal microscopy as described in the legend to Figure 1. (B) N13-P2X7R^High (upper panel) or N13-P2X7R^Low (lower panel) were loaded with MitoTracker Red FM (400 n m) at 37°C for 20 min, then thoroughly rinsed and further incubated at 37°C in complete RPMI medium for 24 h with MP-P2X7R^High released from MitoTracker Green-stained N13-P2X7R^High. At the end of this incubation period cells were analyzed by confocal microscopy. (C) Intracellular ATP content of N13-P2X7R^High or N13-P2X7R^Low after 3 or 24 h (1 d) of co-incubation with MP-P2X7R^High. (D) WB of control or MP-P2X7R^High-pulsed N13-P2X7R^High or N13-P2X7R^Low cell lysates. Actin was used as loading control. (E-G) Densitometric analysis. Isolation of MP-P2X7R^High and co-incubation with either N13-P2X7R^High or N13-P2X7R^Low were performed as described in Materials and Methods. (A) Bar = 20 μm; B, bar = 10 μm. * P < 0.05; ** P < 0.01; *** P < 0.001; ^****  P < 0.0001 by unpaired t test.

Figure 5.

Released mitochondria are taken up by primary microglia in a P2X7R-dependent fashion. Mitotracker Red-labelled microglial cells from P2X7R-WT or P2X7R-KO mice were let adhere to 24-well dishes, rinsed, and further incubated in complete RPMI medium for 24 h with MitoTracker Green FM-labeled MP-P2X7R^High or MP-P2X7R^Low previously isolated from MitoTracker Green FM-labeled source N13 cells (Materials and Methods). At the end of this incubation, the monolayers were rinsed to remove extracellular MPs, fresh medium was added, and the cells analyzed by confocal microscopy as described in the legend to Figure 1. Bars = 5 µm.

Figure 6.

Fusion of MP-P2X7R^High with the recipient cells restores eATP-mediated plasma membrane permeabilization and increases the proliferation rate in a P2X7R-dependent fashion. Fluorescence (A-D, upper panels) or phase contrast (A-D, lower panels) images of eATP-stimulated lucifer yellow uptake of control (A) or MP-P2X7R^High-challenged (B) N13-P2X7R^High, or control (C) or MP-P2X7R^High-challenged (D) N13-P2X7R^Low. Cells were incubated in complete RPMI medium at 37°C in a CO₂ incubator in the absence or presence of MP-P2X7R^High for 24 h, then rinsed, further incubated in complete RPMI at 37°C, and challenged with 3 m m eATP in the presence of 1 mg/mL lucifer yellow. At the end of this incubation, the monolayers were thoroughly rinsed, incubated in fresh complete RPMI medium and analyzed by fluorescence microscopy with a IMT-2 Olympus phase/fluorescence microscope equipped with 20× objective. (E) Lucifer yellow fluorescence was quantitated as described in Materials and Methods. N13-P2X7R^High (F) or N13-P2X7R^Low (G) were seeded at 37°C in a CO₂ incubator in 24-well plastic dishes in complete RPMI medium. After an overnight incubation, a suspension of either MP-P2X7R^High or MP-P2X7R^Low was added to each given cell type, and incubation carried out at 37°C in a CO₂ incubator for further 72 h. Cell number was quantitated by crystal violet (see Materials and Methods). Bars = 50 μm. * P < 0.05; ** P < 0.01; *** P < 0.001; ^****  P < 0.0001 by unpaired t test.

Figure 7.

Microparticle fusion with the recipient cells enhances MGC formation in a P2X7R-dependent fashion. N13-P2X7R^High were incubated in 24-well plastic dishes at 37°C in complete RPMI medium for 48 h as such (A) or in the presence of either MP-P2X7R^Low (B) or MP-P2X7R^High (C). Time course of MGC formation shown as fusion index in N13-P2X7R^High monolayers incubated in the presence of MP-P2X7R^Low, MP-P2X7R^High, or left unchallenged (D). N13-P2X7R^Low were incubated in 24-well plastic dishes at 37°C in complete RPMI medium for 48 h as such (E) or in the presence of either MP-P2X7R^Low (F) or MP-P2X7R^High (G). Time course of MGC formation in N13-P2X7R^Low monolayers incubated in the presence of MP-P2X7R^Low, MP-P2X7R^High, or left unchallenged (H). Fusion index was calculated as follows: (number of nuclei within MGC/total number of nuclei) × 100. All pictures were taken with an Olympus microscope, as described in the legend to Figure 6.Bars = 10 µm. ** P < 0.01; *** P < 0.001; ^****  P < 0.0001 by unpaired t test.