INTRODUCTION 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. However, mitochondria and its typical hallmarks (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 isolation kits are available commercially, they can be somewhat expensive and not suitable for some downstream applications. In this project, we provide an alternative purification method yielding mitochondria of high purity and integrity using human platelets. OBJECTIVES Evaluate the purity, integrity and yield of two different methods of isolation of mitochondria in human platelets. METHODS First, platelets were isolated from the blood of healthy donors. Then the brute fraction of platelet‐derived mitochondria was obtained using a potter homogenizer, followed by several differential centrifugation. To obtain the purified fraction, the mitochondrial extract was centrifuged on a discontinuous Percoll gradient (GE Healthcare). The purity of mitochondria was determined by flow cytometry (FC500, Beckman Coulter) using specific platelet marker anti‐CD41‐FITC (BioLegend), and by transmission electron microscopy (TEM). The respiratory capacity of mitochondria was measured by high‐resolution respirometry (Oroboros instruments). The total yield of mitochondria was determined by flow cytometry using MitoTracker Deep Red (Molecular Probes) and by the micro‐Smith method. Finally, the integrity of the mitochondrial membrane potential was assessed with JC‐1 staining (Molecular Probes). RESULTS Data generated by flow cytometry shows that the Percoll gradient significantly purified mitochondria by removing 50% of platelet membrane debris (paired t‐test, p < 0.01). TEM analysis shows similar results. Mitochondrial respiration following the substrate uncouple inhibitor titration protocol is identical in purified and in brute mitochondria. Additionally, the cytochrome c effect is 5%, while JC‐1 staining shows no significant difference between methods suggesting integrity both in the inner and outer mitochondrial membrane. On the other hand, the mitochondrial protein yield was significantly decreased after purification (paired t‐test, p < 0.01). CONCLUSIONS Results of this study suggest that the Percoll discontinuous gradient purifies viable platelet‐derived mitochondria. Conversely, mitochondrial yield may be less important than obtained in other methods; however, it could be explained by the clustering of mitochondria containing less platelet debris. Relatively inexpensive, this method of purification is ideal for studying the downstream effects of intact mitochondria in mitochondrial‐related disea...

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 (IC 50 5 1.53 mM) 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.

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.

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INTRODUCTIONThe 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. However, mitochondria and its typical hallmarks (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 isolation kits are available commercially, they can be somewhat expensive and not suitable for some downstream applications. In this project, we provide an alternative purification method yielding mitochondria of high purity and integrity using human platelets.OBJECTIVESEvaluate the purity, integrity and yield of two different methods of isolation of mitochondria in human platelets.METHODSFirst, platelets were isolated from the blood of healthy donors. Then the brute fraction of platelet‐derived mitochondria was obtained using a potter homogenizer, followed by several differential centrifugation. To obtain the purified fraction, the mitochondrial extract was centrifuged on a discontinuous Percoll gradient (GE Healthcare). The purity of mitochondria was determined by flow cytometry (FC500, Beckman Coulter) using specific platelet marker anti‐CD41‐FITC (BioLegend), and by transmission electron microscopy (TEM). The respiratory capacity of mitochondria was measured by high‐resolution respirometry (Oroboros instruments). The total yield of mitochondria was determined by flow cytometry using MitoTracker Deep Red (Molecular Probes) and by the micro‐Smith method. Finally, the integrity of the mitochondrial membrane potential was assessed with JC‐1 staining (Molecular Probes).RESULTSData generated by flow cytometry shows that the Percoll gradient significantly purified mitochondria by removing 50% of platelet membrane debris (paired t‐test, p < 0.01). TEM analysis shows similar results. Mitochondrial respiration following the substrate uncouple inhibitor titration protocol is identical in purified and in brute mitochondria. Additionally, the cytochrome c effect is 5%, while JC‐1 staining shows no significant difference between methods suggesting integrity both in the inner and outer mitochondrial membrane. On the other hand, the mitochondrial protein yield was significantly decreased after purification (paired t‐test, p < 0.01).CONCLUSIONSResults of this study suggest that the Percoll discontinuous gradient purifies viable platelet‐derived mitochondria. Conversely, mitochondrial yield may be less important than obtained in other methods; however, it could be explained by the clustering of mitochondria containing less platelet debris. Relatively inexpensive, this method of purification is ideal for studying the downstream effects of intact mitochondria in mitochondrial‐related diseases.Support or Funding InformationCanadian Institutes of Health Research, New Brunswick Health Research Foundation, New Brunswick Innovation FoundationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

INTRODUCTIONNeutrophils play a key role as the first line of defense against pathogens. Their ability to generate and release inflammatory mediators (eicosanoids or cytokines) helps promote the inflammatory response and consequently restore the hemostasis. While 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 an important therapeutic approach pursued by numerous researchers. The neutrophil's lifespan is relatively short (8–12h), which can be problematic for some in vitro experiments. To address this issue, researchers used the human monomyelocyte cell line PLB‐985 as an alternative for some of their in vitro experiments. PLB‐985 cells can be differentiated into a more neutrophil‐like phenotype upon exposure to several agonists, including dimethyl sulfoxide (DMSO). Whether this differentiation of PLB‐985 affects important features related to neutrophil's normal functions (i.e. mitochondrial activity, eicosanoid production) remains elusive and characterizing these changes will be the focal point of this project.OBJECTIVESWe assessed and compared the mitochondrial activity and inflammatory state of PLB‐985 cells following differentiation.METHODSDifferentiation of PLB‐985 cells was performed using 1% DMSO for 6 days and was confirmed by immunofluorescence microscopy and flow cytometry using an anti‐CD11b‐PE cell surface antibody. Proliferation and apoptosis assays were performed by flow cytometry using the eFluor 670 and 7‐aminoactinomycin D labelers respectively. The mitochondrial respiratory assay was determined by high‐resolution O2 respirometry (Oroboros Instrument). The inflammatory state of the cells was evaluated by transcriptomic, proteomic and lipidomic approaches.RESULTSDifferentiation affected the proliferation of PLB‐985 cells (P<0.0001), without inducing apoptosis. A significant decrease in the mitochondrial respiration was observed in differentiated PLB‐985 cells (P<0.0005). However, the overall mitochondria content was not affected. Immunoblotting with mitochondrial antibodies revealed a strong modulation of the succinate dehydrogenase A (P<0.0001), superoxide dismutase 2 (P<0.0001), ubiquinol‐cytochrome c reductase core protein 2 (P<0.05) and ATP synthase subunit α (P<0.01) in differentiated PLB‐985 cells. Finally, eicosanoids (leukotriene B4, 12‐hydroxyheptadecatrienoic and 15‐hydroxyeicosatetraenoic acids) production was significantly increased in differentiated cells (P<0.01).CONCLUSIONSOur 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 modulations of the inflammatory state of the cell. Finally, our data suggests that PLB‐985 cells could be a suitable in vitro candidate to study mitochondrial‐related dysfunctions in inflammatory diseases.Support or Funding InformationCIHR, NBHRF, NBIFThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

IntroductionEicosanoids are lipid mediators involved in several critical steps of the inflammatory response. While the response is necessary for the host's defense against pathogens, uncontrolled or unregulated production of these mediators has been associated with several types of chronic inflammatory diseases, including arthritis, asthma and cardiovascular diseases. Since chronic inflammation is the foundation and a key manifestation for various inflammatory diseases, it is not surprising that enzymes implicated in the production of eicosanoids have been strategically targeted for potential therapeutic approaches. The 12‐hydroxyeicosatetraenoic acid (12(S)‐HETE) lipid mediator is among those inflammatory molecules abundantly produce in various diseases and is primarily biosynthesized via the 12(S)‐lipoxygenase (12‐LO) pathway. The abundance of 12(S)‐HETE and its contribution to several chronic inflammatory diseases, such as arthritis or cancer, has been well established over the last few years. While most developed compounds primarily target the 5‐lipoxygenase (5‐LO) or the cyclooxygenase (COX) pathways, very few compounds have been designed to selectively inhibit the 12‐LO pathway. Given the physiological importance of 12(S)‐HETE in inflammatory diseases, it is somewhat surprising that the enzyme has not been the focus of more pharmacological targeting. Amongst the compounds that have generated interest in the field of eicosanoids are natural‐occurring flavonoids, a class of bioactive plant compounds. Flavonoids exhibit several beneficial properties including anti‐inflammatory and anti‐oxidant capacities and can impact on some cardiovascular risk factors. In this study, we examined whether the distribution of hydroxyl groups among flavones could influence their potency as 12‐LO inhibitors.Research approachPlatelets isolated from healthy consenting volunteer were pre‐incubated in presence of the test compounds or vehicle and then stimulated with thrombin to initiate 12(S)‐HETE production. The 12(S)‐HETE was quantified by high‐performance liquid chromatography with UV detection. Platelet activation was accessed by CD62P externalization (P‐Selectin) using flow cytometry. Finally, the effects of the compounds on the cell's metabolism were evaluated by high‐resolution respiratory assays (Oroboros Instrument). This study was approved by Université de Moncton review committee for research involving human subjects.ResultsWe demonstrated that the peracetylated quercetin (1 μM) inhibits 12(S)‐HETE production by 39.2% ± 7.42 (mean±SEM). For comparison, baicalein the most commonly used 12‐LO inhibitor, had very similar inhibitory potency as it reduced 12(S)‐HETE production by 39.4 ± 4.57% (mean±SEM). Peracetylated quercetin and baicalein had IC50 values of 1.62 and 1.22 μM respectively. However, peracetylated quercetin demonstrated selectivity for the 12‐LO, which was not the case for baicalein when compounds where tested for COX‐1 inhibition. Finally, the quercetin derivative did not exhibit any off‐target effects on platelet activation (P‐Selectin expression) or metabolism (electron transfer chain).ConclusionThis study characterizes the peracetylated quercetin as a more selective platelet‐type 12‐LO inhibitor than baicalein, with no measurable undesirable effects on other cellular pathways.Support or Funding InformationCanadian Institutes of Health Research (LHB, JLJ, MES), New Brunswick Innovation Foundation (LHB, MES), New Brunswick Health Research Foundation (JLJ, JLL), Natural Sciences and Engineering Research Council of Canada (NP, MT)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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