Platelets release mitochondria serving as substrate for bactericidal group IIA-secreted phospholipase A2 to promote inflammation

Abstract

Mitochondrial DNA (mtDNA) is a highly potent inflammatory trigger and is reportedly found outside the cells in blood in various pathologies. Platelets are abundant in blood where they promote hemostasis. Although lacking a nucleus, platelets contain functional mitochondria. On activation, platelets produce extracellular vesicles known as microparticles. We hypothesized that activated platelets could also release their mitochondria. We show that activated platelets release respiratory-competent mitochondria, both within membrane-encapsulated microparticles and as free organelles. Extracellular mitochondria are found in platelet concentrates used for transfusion and are present at higher levels in those that induced acute reactions (febrile nonhemolytic reactions, skin manifestations, and cardiovascular events) in transfused patients. We establish that the mitochondrion is an endogenous substrate of secreted phospholipase A2 IIA (sPLA2-IIA), a phospholipase otherwise specific for bacteria, likely reflecting the ancestral proteobacteria origin of mitochondria. The hydrolysis of the mitochondrial membrane by sPLA2-IIA yields inflammatory mediators (ie, lysophospholipids, fatty acids, and mtDNA) that promote leukocyte activation. Two-photon microscopy in live transfused animals revealed that extracellular mitochondria interact with neutrophils in vivo, triggering neutrophil adhesion to the endothelial wall. Our findings identify extracellular mitochondria, produced by platelets, at the midpoint of a potent mechanism leading to inflammatory responses.

Figure 1

Mitochondrial distribution within resting platelets. Mitochondria (black arrows) in resting platelets examined by (A) TEM and (B) confocal scanning laser microscopy (CSLM). (C) Mitochondria are located proximally to the platelet plasma membrane (n = 31; data represent the mean ± standard error of the mean [SEM], ***P < .0001, Student t test).

Figure 2

Activated platelets release extracellular mitochondria. (A) Platelet-free supernatants resulting from the isolation of thrombin-activated platelets consume O₂ via the electron transport chain following cell permeabilization with saponin detergent (50 μg/mL). No O₂ consumption is detected in supernatants obtained from resting platelets (n = 4; data are mean ± SEM). (B) Three predicted types of extracellular microparticles (MPs) produced on platelet activation: mitochondria (freeMitos), mitochondria-containing MPs (mitoMPs), and MPs lacking mitochondria (MPs). (C) Isolation of freeMitos using anti-TOM22 microbeads (or IgG control) in thrombin-stimulated platelets and mtDNA quantification (n = 4; data are mean ± SEM, **P < .005, Student t test). (D) TEM visualization of freeMitos (white arrows), mitoMPs (black arrows), and MPs (black arrowheads) released from thrombin-activated platelets. (E) Three-dimensional CSLM reconstruction of the supernatant of thrombin-activated platelets. Populations represented in image are platelets (black arrow), MPs (white arrows), mitoMPs (white arrowheads), and freeMitos (black arrowheads). (F) High-sensitivity flow cytometry (hs-FCM) analysis of resting platelets (upper panel, top right quadrant) and thrombin-activated platelets, which show 3 additional, distinct populations of particles, ie, freeMitos (bottom panel, top left quadrant, blue), mitoMPs (bottom panel, top right quadrant, pink), and mitochondria-free MPs (bottom panel, bottom right quadrant, red). Bottom left quadrant of both upper and lower panels represents background noise (gray). FSC-PMT and SSC dot plots of platelets (first right panel) and 3 populations of microparticles: freeMitos (second right panel), mitoMPs (third right panel), and MPs (fourth right panel). The relative diameters are presented according to size-defined microsphere calibrations. (G) Release of (left) freeMito, (center) mitoMPs, and (right) MPs from thrombin-activated platelets require intact actin microfilament dynamics. Mitochondrial release is significantly reduced on addition of actin inhibitors (cytochalasin [B,D-E] and latrunculin [A]), but not tubulin polymerization inhibitor (nocodazole) (n = 4; data are mean ± SEM, *P < .05, **P < .005, and ***P < .001, Student t test). (H) Heat-aggregated IgG (HA-IgG), thrombin, collagen, cross-linked collagen related peptide (CRP-XL), and phorbol 12-myristate 13-acetate (PMA) trigger the release of (left) extracellular freeMitos, (center) mitoMPs, and (right) MPs quantified by hs-FCM (n = 4; data are mean ± SEM. *P < .05, **P < .005, and ***P < .001 vs supernatant from resting platelets, Student t test).

Figure 3

Extracellular mitochondria are present in various situations where platelets are known to be activated. (A) Platelet mitoMPs (CD41⁺MitoTracker⁺) are found in higher concentrations in the synovial fluid of RA patients (n = 20) than in the synovial fluid of osteoarthritis patients (OA, n = 14; data are mean ± SEM, *P < .05, Mann-Whitney test). (B) FreeMitos are detected in fresh SF of RA patients. Isolation of freeMitos in RA SF (from 3 different patients) with anti-TOM22 microbeads (or control IgG; supplemental Figure 3) and mtDNA quantification. (C) TEM imaging of (left) a freeMito and (right) a mitoMP from fresh RA SF. (D) O₂ consumption is observed in PFP obtained at the indicated time intervals from platelet storage bags. (E) Isolation of freeMitos (supplemental Figure 3) in PFP along with mtDNA quantification reveals an abundance of freeMito at day 5 (n = 6; data are mean ± SEM, *P < .05 vs day 0, paired Student t test). (F) High-sensitivity flow cytometry (hs-FCM) analysis of resting platelets (upper panel, top right quadrant) and thrombin-activated platelets, which show 3 additional distinct populations of particles, ie, freeMitos (bottom panel, top left quadrant, blue), mitoMPs (bottom panel, top right quadrant, pink), and mitochondria-free MPs (bottom panel, bottom right quadrant, red). Bottom left quadrant of both upper and lower panels represents background noise (gray). FSC-PMT and SSC dot plots of platelets (first right panel) and 3 populations of microparticles: freeMitos (second right panel), mitoMPs (third right panel), and MPs (fourth right panel). The relative diameters are presented according to size-defined microsphere calibrations. (G) TEM imaging of PFP collected on day 5 confirming the presence of (left) freeMitos and (right) mitoMPs. (H) Mitochondrial membrane potential is detected in freeMitos and mitoMPs collected from PFP, as measured by a JC-1 assay using hs-FCM (red to green ratio) (n = 5; data are mean ± SEM). (I) Extracellular mitochondria (as detected by mtDNA quantification) are found at higher concentration in PFP of platelet storage bags that have cause adverse transfusion reaction to the recipient (no adverse reaction group [n = 61] vs adverse reaction group [n = 74] matched in term of storage duration; data are mean ± SEM, ***P < .001, Student t test). Adverse reaction measured include mainly febrile nonhemolytic reactions, skin manifestations such as itching or skin rash, and cardiovascular events such as hypotension or tachycardia.

Figure 4

The mitochondrion is a substrate for the bactericidal sPLA₂-IIA. (A) Quantification of sPLA₂-IIA in human platelets by time-resolved immunofluorescence (n = 3; data are mean ± SEM). (B) sPLA₂-IIA immunoblotting of mitochondria isolated with anti-TOM22 microbeads reveals binding of human recombinant sPLA₂-IIA to mitochondria (supplemental Figure 3). (C) Mitochondria were incubated in (left) the absence or (right) presence of Alexa488-conjugated sPLA₂-IIA and analyzed by hs-FCM. The significant shift in the fluorescent population size (right) indicates that sPLA₂-IIA binds mitochondria. (D-E) Catalytic activity of human recombinant sPLA₂-IIA (or PBS as vehicle) toward mitochondria. Mitochondrial membrane phospholipid hydrolysis by sPLA₂-IIA yields (D) lysophospholipids and (E) fatty acids as quantified by mass spectrometry. (F) sPLA₂-IIA affects mitochondrial structural integrity. Scanning electronic micrographs of mitochondria incubated in the (left) absence or (right) presence of human recombinant sPLA₂-IIA. (G) Mitochondria (magenta) release mtDNA (blue) on incubation with recombinant sPLA₂-IIA (upper panels). Extracellular mtDNA accumulation (arrow) is apparent in the presence of sPLA₂-IIA. Differential interference contrast images are shown for reference (lower panels). (H) mtDNA extrusion is amplified in presence of human recombinant sPLA₂-IIA (0.2 μg/mL, 30 minutes at 37°C), as quantified by Sytox Green nucleic acid stain assay (n = 6; data are mean ± SEM, *P < .05, Student t test).

Figure 5

Extracellular mitochondria interact with neutrophils. (A) Intravenously injected fluorescence-labeled mitochondria (MitoTracker Deep Red) associate with mouse neutrophils (Gr1⁺ cells) in vivo as measured by flow cytometry (n = 6; data are mean ± SEM, ***P < .001, Student t test). (B) Intravenous injection of mitochondria induces neutrophil rolling in LysM-eGFP mice. (Center and right) Neutrophil (green) velocity is significantly reduced (n = 89; supplemental Movie 3) in blood (red) following intravenous injection of mitochondria compared with (left) Tyrode buffer as vehicle (n = 51; data are mean ± SEM, ***P < .001, Student t test) (C) Scanning electronic micrographs of mitochondria in association with (left) freshly isolated human neutrophil and (right) ensuing neutrophil structural change (29.2 ± 2.11%, n = 3) after a 30-minute incubation in the presence of human recombinant sPLA₂-IIA.

Figure 6

Interaction of human neutrophils with the mitochondria/sPLA₂-IIA complex promotes the release of proinflammatory mediators. (A) Human neutrophils associate with the mitochondria/sPLA₂-IIA complex in vitro as measured by flow cytometry analysis of human neutrophils incubated with fluorescently labeled mitochondria (MitoTracker Deep Red) in the (left) absence or (right) presence of Alexa488-conjugated sPLA₂-IIA. (B) Three-dimensional CSLM reconstruction of mitochondria (red) and sPLA₂-IIA (green) colocalizing within neutrophils (blue nuclei, Hoechst stain; gray cytoplasm, CellTracker CMTPX). (C) Mitochondria are internalized in human neutrophils via an endocytosis-dependent pathway. Graph bars representation of the relative localization (surface vs intracellular) of the mitochondria inside neutrophils following pretreatment with indicated inhibitors (nystatin for inhibition of caveolin-mediated endocytosis; chlorpromazine for inhibition of clathrin-mediated endocytosis; dynasore for inhibition of dynamin-mediated endocytosis; nocodazole for inhibition of polymerization of microtubule [endocytosis and phagocytosis]; cytochalasin B for inhibition of polymerization of actin [endocytosis and phagocytosis]). Data were obtained from 100 neutrophils per condition repeated 3 times (n = 3, *P < .01, **P < .001, and ***P < .0001, Mann-Whitney test compare with diluent). (D) Mitochondrial hydrolytic products derived from the action of the mitochondria/sPLA₂-IIA complex (Figure 4D-E) induce proinflammatory responses in human neutrophils. The total 5-lipoxygenase products (5-LO products) were quantified by high-performance liquid chromatography (n = 4; data are mean ± SEM, **P < .005 vs control, Student t test). (E) The freeMito fraction induces NET formation in vitro and is enhanced by sPLA₂-IIA. NET formation (left panel, DNA, blue, white dotted line) was confirmed by confocal imaging after treatment of mitochondria (red, right panel) with sPLA₂-IIA. sPLA₂-IIA significantly enhances NET formation by mitochondria (upper right panel, n ≥ 7; data are mean ± SEM, *P < .05 and **P < .005, Student t test). Hydrolysis products from mitochondria/sPLA2-IIA complex activity also induce significant NET formation (lower right panel, n ≥ 3; data are mean ± SEM, **P < .005 and ***P < .001, Student t test).

Figure 7

Extracellular mitochondria and sPLA₂-IIA amplify inflammation in vivo. (A) Intravenous injection of mitochondrial hydrolytic products (sPLA₂-IIA–treated mitochondria, black triangle) in sPLA₂-IIA–deficient mice significant lowers body temperature (Δ temperature vs PBS-injected mice of respective background) after 4 hours (n = 6/group; data are mean ± SEM, **P < .005 compared with sPLA₂-IIA–untreated mitochondria [▪] or sPLA₂-IIA alone [●]). Intravenous injection of mitochondria (sPLA₂-IIA untreated, ☐) in sPLA₂-IIA–sufficient mice significantly lowers body temperature after 24 hours. Only a modest temperature decrease was observed in sPLA₂-IIA–untreated mitochondria (▪) in sPLA₂-IIA–deficient mice (n ≥ 3/group; data are mean ± SEM, **P < .005). (B) sPLA₂-IIA–generated mitochondrial products trigger inflammation in vivo. Mitochondria incubated in the presence of recombinant sPLA₂-IIA and injected into the air pouch of C57BL/6N mice induce the production of (left) IL-1β and (right) IL-6. Diluent (PBS), sPLA₂-IIA alone, or untreated mitochondria induce modest cytokine production when injected separately (n = 7; data are mean ± SEM, **P < .005 compared with mitochondria incubated in the absence of sPLA₂-IIA). (C) Mitochondria accumulation in the liver induces numerous proinflammatory genes that are amplified in the presence of endogenous sPLA₂-IIA. mRNA expression of inflammatory genes relevant to neutrophil function was quantified in the liver of sPLA₂-IIA–sufficient and –deficient mice intravenously injected with mitochondria (n = 3 per group; data expressed as the ratio of specific mRNA expression ratio (sPLA₂-IIA sufficient/deficient mice). (D) Schematic representation of the mechanism of action of extracellular mitochondria and sPLA₂-IIA in sterile inflammatory conditions. On activation, platelets release MPs, mitoMPs, and freeMitos. Mitochondrial membrane phospholipids may be hydrolyzed by sPLA₂-IIA, generating bioactive mediators (fatty acids, lysophospholipids, and mtDNA) and promoting neutrophil proinflammatory responses.