Functional platelet-derived mitochondria induce the release of human neutrophil microvesicles.

Inflammation is an essential process of host defense against infections, illness, or tissue damage. Polymorphonuclear neutrophils (PMN) are among the first immune cells involved in acute inflammatory responses and are on the front line in the fight against bacterial infections. In the presence of bacterial fragments, PMN release inflammatory mediators, enzymes, and microvesicles in the extracellular milieu to recruit additional immune cells required to eliminate the pathogens. Recent evidence shows that platelets (PLTs), initially described for their role in coagulation, are involved in inflammatory responses. Furthermore, upon activation, PLT also release functional mitochondria (freeMitos) within their extracellular milieu. Mitochondria share characteristics with bacterial and mitochondrial damage-associated molecular patterns, which are important contributors in sterile inflammation processes. Deep sequencing transcriptome analysis demonstrates that freeMitos increase the mitochondrial gene expression in PMN. However, freeMitos do not affect the mitochondrial-dependent increase in oxygen consumption in PMN. Interestingly, freeMitos significantly induce the release of PMN-derived microvesicles. This study provides new insight into the role of freeMitos in the context of sterile inflammation.

Rapid isolation and purification of functional platelet mitochondria using a discontinuous Percoll gradient.

The isolation of mitochondria is gaining importance in experimental and clinical laboratory settings. The mitochondrion is known as the powerhouse of the cell as it produces the energy to power most cellular functions but is also involved in many cellular processes. Of interest, mitochondria and mitochondrial components (i.e. circular DNA, N-formylated peptides, cardiolipin) have been involved in several human inflammatory pathologies, such as cancer, Alzheimer's disease, Parkinson's disease, and rheumatoid arthritis. Therefore, stringent methods of isolation and purification of mitochondria are of the utmost importance in assessing mitochondrial-related diseases. While several mitochondrial isolation methods have been previously published, these techniques are aimed at yielding mitochondria from cells types other than platelets. In addition, little information is known on the number of platelet-derived microparticles that can contaminate the mitochondrial preparation or even the overall quality and integrity of the mitochondria. In this project, we provide an alternate purification method yielding mitochondria of high purity and integrity from human platelets. Using human platelets, flow cytometry and transmission electron microscopy experiments were performed to demonstrate that the Percoll gradient method yielded significantly purified mitochondria by removing platelet membrane debris. Mitochondrial respiration following the substrate-uncoupler-inhibitor-titration (SUIT) protocol was similar in both the purified and crude mitochondrial extraction methods. Finally, the cytochrome c effect and JC-1 staining did not exhibit a significant difference between the two methods, suggesting that the mitochondrial integrity was not affected. Our study suggests that the Percoll discontinuous gradient purifies viable platelet-derived mitochondria by removing platelet-derived debris, including microparticles, therefore confirming that this isolation method is ideal for studying the downstream effects of intact mitochondria in mitochondrial-related diseases.

Identification of Peracetylated Quercetin as a Selective 12-Lipoxygenase Pathway Inhibitor in Human Platelets.

The inflammatory response is necessary for the host's defense against pathogens; however, uncontrolled or unregulated production of eicosanoids has been associated with several types of chronic inflammatory diseases. Thus, it is not surprising that enzymes implicated in the production of eicosanoids have been strategically targeted for potential therapeutic approaches. The 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE] lipid mediator is among inflammatory molecules that are abundantly produced in various diseases and is primarily biosynthesized via the 12(S)-lipoxygenase pathway. The effects of the abundance of 12(S)-HETE and its contribution to several chronic inflammatory diseases have been well studied over the last few years. While most developed compounds primarily target the 5-lipoxygenase (5-LO) or the cyclooxygenase (COX) pathways, very few compounds selectively inhibiting the 12-lipoxygenase (12-LO) pathway are known. In this study, we examined whether the distribution of hydroxyl groups among flavones could influence their potency as 12-LO inhibitors. Using human platelets, the human embryonic kidney 293 (HEK293) cell line expressing 5-LO, and human polymorphonuclear leukocytes (PMNLs) we investigated the effects of these compounds on several inflammatory pathways, namely, 12-LO, 5-LO, and COX. Using high-resolution respirometry and flow cytometry, we also evaluated some normal cell functions that could be modulated by our compounds. We identified a peracetylated quercetin (compound 6) that exerts potent inhibitory activity toward the platelet 12-LO pathway (IC50 = 1.53 µM) while having a lesser affinity toward the COX pathway. This study characterizes the peracetylated quercetin (compound 6) as a more selective platelet-type 12-LO inhibitor than baicalein, with no measurable nontargeted effects on the platelet's activation or overall cell's oxygen consumption.

Purification of Functional Platelet Mitochondria Using a Discontinuous Percoll Gradient.

The isolation of mitochondria is gaining importance in experimental and clinical laboratory settings. Of interest, mitochondria and mitochondrial components (i.e., circular mitochondrial DNA, N-formylated peptides, cardiolipin) have been involved in several human inflammatory pathologies, such as cancer, Alzheimer's disease, Parkinson's disease, and rheumatoid arthritis. While several mitochondrial isolation methods have been previously published, these techniques are aimed at yielding mitochondria from cell types other than platelets. In addition, little information is known on the number of platelet-derived microvesicles that can contaminate the mitochondrial preparation or even the overall quality as well as functional and structural integrity of mitochondria. Here we describe a purification method, using a discontinuous Percoll gradient, yielding mitochondria of high purity and integrity from human platelets.

Evaluating the mitochondrial activity and inflammatory state of dimethyl sulfoxide differentiated PLB-985 cells.

Neutrophils play a key role in the innate immunity with their ability to generate and release inflammatory mediators that promote the inflammatory response and consequently restore the hemostasis. As active participants in several steps of the normal inflammatory response, neutrophils are also involved in chronic inflammatory diseases such as asthma, atherosclerosis, and arthritis. Given their dual role in the modulation of inflammation, regulating the inflammatory response of neutrophils has been suggested as an important therapeutic approach by numerous researchers. The neutrophils have a relatively short lifespan, which can be problematic for some in vitro experiments. To address this issue, researchers have used the human monomyelocyte cell line PLB-985 as an in vitro model for exploratory experiments addressing neutrophil-related physiological functions. PLB-985 cells can be differentiated into a neutrophil-like phenotype upon exposure to several agonists, including dimethyl sulfoxide (DMSO). Whether this differentiation of PLB-985 affects important features related to the neutrophil's normal functions (i.e., mitochondrial activity, eicosanoid production) remains elusive, and characterizing these changes will be the focal point of this study. Our results indicate that the differentiation affected the proliferation of PLB-985 cells, without inducing apoptosis. A significant decrease in mitochondrial respiration was observed in differentiated PLB-985 cells. However, the overall mitochondria content was not affected. Immunoblotting with mitochondrial antibodies revealed a strong modulation of the succinate dehydrogenase A, superoxide dismutase 2, ubiquinol-cytochrome c reductase core protein 2 and ATP synthase subunit α in differentiated PLB-985 cells. Finally, eicosanoids (leukotriene B4, 12-hydroxyheptadecatrienoic and 15-hydroxyeicosatetraenoic acids) production was significantly increased in differentiated cells. In summary, our data demonstrate that the differentiation process of PLB-985 cells does not impact their viability despite a reduced respiratory state of the cells. This process is also accompanied by modulation of the inflammatory state of the cell. Of importance, our data suggest that PLB-985 cells could be suitable in vitro candidates to study mitochondrial-related dysfunctions in inflammatory diseases.