Luc H. Boudreau

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

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.

The exposure of autoantigens by microparticles underlies the formation of potent inflammatory components: the microparticle‐associated immune complexes

Immunoglobulins, antigens and complement can assemble to form immune complexes (IC). ICs can be detrimental as they propagate inflammation in autoimmune diseases. Like ICs, submicron extracellular vesicles termed microparticles (MP) are present in the synovial fluid from patients affected with autoimmune arthritis. We examined MPs in rheumatoid arthritis (RA) using high sensitivity flow cytometry and electron microscopy. We find that the MPs in RA synovial fluid are highly heterogeneous in size. The observed larger MPs were in fact MP‐containing ICs (mpICs) and account for the majority of the detectable ICs. These mpICs frequently express the integrin CD41, consistent with platelet origin. Despite expression of the Fc receptor FcγRIIa by platelet‐derived MPs, we find that the mpICs form independently of this receptor. Rather, mpICs display autoantigens vimentin and fibrinogen, and recognition of these targets by anti‐citrullinated peptide antibodies contributes to the production of mpICs. Functionally, platelet mpICs are highly pro‐inflammatory, eliciting leukotriene production by neutrophils. Taken together, our data suggest a unique role for platelet MPs as autoantigen‐expressing elements capable of perpetuating formation of inflammatory ICs.

Platelet microparticles are internalized in neutrophils via the concerted activity of 12-lipoxygenase and secreted phospholipase A2-IIA

Significance On activation, blood platelets package components from their cytoplasm into microparticles (MPs), tiny vesicles released by cytoplasmic membrane budding and shedding. Given that MPs can impact other cellular lineages on internalization, we aimed to decipher the mechanisms promoting MP internalization by cellular recipients. We modeled MP internalization by neutrophils and identified a predominant lipid, 12(S)-hydroxyeicosatetranoic acid, as a mediator critical for the promotion of MP internalization. MPs were found inside neutrophils from individuals with rheumatoid arthritis, and their presence in neutrophils in the joints of mice treated with arthritogenic serum is dependent on the expression of enzymes implicated in the generation of 12(S)-hydroxyeicosatetranoic acid. These findings reveal a unique molecular mechanism implicated in MP internalization relevant to inflammatory processes. Platelets are anucleated blood elements highly potent at generating extracellular vesicles (EVs) called microparticles (MPs). Whereas EVs are accepted as an important means of intercellular communication, the mechanisms underlying platelet MP internalization in recipient cells are poorly understood. Our lipidomic analyses identified 12(S)-hydroxyeicosatetranoic acid [12(S)-HETE] as the predominant eicosanoid generated by MPs. Mechanistically, 12(S)-HETE is produced through the concerted activity of secreted phospholipase A2 IIA (sPLA2-IIA), present in inflammatory fluids, and platelet-type 12-lipoxygenase (12-LO), expressed by platelet MPs. Platelet MPs convey an elaborate set of transcription factors and nucleic acids, and contain mitochondria. We observed that MPs and their cargo are internalized by activated neutrophils in the endomembrane system via 12(S)-HETE. Platelet MPs are found inside neutrophils isolated from the joints of arthritic patients, and are found in neutrophils only in the presence of sPLA2-IIA and 12-LO in an in vivo model of autoimmune inflammatory arthritis. Using a combination of genetically modified mice, we show that the coordinated action of sPLA2-IIA and 12-LO promotes inflammatory arthritis. These findings identify 12(S)-HETE as a trigger of platelet MP internalization by neutrophils, a mechanism highly relevant to inflammatory processes. Because sPLA2-IIA is induced during inflammation, and 12-LO expression is restricted mainly to platelets, these observations demonstrate that platelet MPs promote their internalization in recipient cells through highly regulated mechanisms.

Synthesis and 5-lipoxygenase inhibitory activity of new cinnamoyl and caffeoylclusters.

Novel cinnamoyl and caffeoyl clusters were synthesized by multiple Cu(I)-catalyzed [1,3]-dipolar cycloadditions and their anti-5-lipoxygenase inhibitory activity was tested. Caffeoyl cluster showed an improved 5-lipoxygenase inhibitory activity compared to caffeic acid, with caffeoyl trimer 16 and tetramer 19 showing the best 5-lipoxygenase inhibitory activity.

Novel 5-lipoxygenase isoforms affect the biosynthesis of 5-lipoxygenase products

5‐Lipoxygenase (5‐LO) is the essential enzyme for the biosynthesis of leukotrienes, important mediators of inflammation. This study investigated whether variants of 5‐LO exist in human leukocytes. 5‐LO mRNA isoforms that are consistent with alternative splicing were identified by RT‐PCR in a cell line or cell type‐specific pattern. All evaluated cells expressed mRNA containing all 14 exons of 5‐LO with the expected splicing sites. Individual isoforms that retained intron 10 (α‐10), lacked exon 13 (Δ‐13), and lacked exons 10 and 13 (Δ‐10,13) or that lacked the first 96 base pairs of exon 10 (Δ‐p10) were identified. Immunoreactive bands coeluting with the cloned α‐10 and Δ‐13 isoforms were measured in primary neutrophils and in Raji cells. When expressed in HEK293 cells, alternative proteins were without catalytic activity. However, when coexpressed with the active full‐length 5‐LO, alternative isoforms significantly decreased the biosynthesis of 5‐LO products by up to 44%, as assessed by reverse‐phase HPLC analysis. Additionally, in stimulated neutrophils the full‐length active 5‐LO was detected by immunoblot in both nuclear and non‐nuclear compartments, while the Δ‐13 isoform was only detected in the nuclear fraction. These alternative 5‐LO isoforms may represent a new mechanism for the regulation of the 5‐LO pathway and lipid mediator biosynthesis.—Boudreau, L. H., Bertin, J., Robichaud, P. P., Laflamme, M., Ouellette, R. J., Flamand, N., Surette, M. E. Novel 5‐lipoxygenase isoforms affect the biosynthesis of 5‐lipoxygenase products. FASEB J. 25, 1097–1105 (2011). www.fasebj.org

Caffeoyl and cinnamoyl clusters with anti-inflammatory and anti-cancer effects. Synthesis and structure–activity relationship

The syntheses of twelve caffeoyl/cinnamoyl clusters and their anti-inflammatory and anti-cancer effects are described. Synthesis of the title compounds involved a multiple copper(I)-catalyzed Huisgen 1,3-dipolar cycloaddition. Azide or alkyne functionalized cinnamoyl or caffeoyl moieties are attached to the selected core molecules to allow variation of the introduced cinnamoyl or caffeoyl moieties in order to compare their effects on 5-lipoxygenase (5-LO) inhibition and on cell proliferation in cancerous (MCF7) and non cancerous (MCF10A) human mammary epithelial cell lines. Caffeoyl dimer 13, trimer 17, and tetramer 19, inhibited 5-LO product synthesis in a cell-free assay with IC50 values ranging from 0.66 to 0.79 μM. These compounds surpassed the inhibitory activity of caffeic acid by more than 10-fold. Monomer 11 caused almost 95% inhibition of 5-LO and surpassed the known 5-LO inhibitor zileuton in a cell-based assay. Trimer compounds 15, 17 and tetramer 19 decreased proliferation rates of MCF-7 cells by 36, 23 and 47%, respectively, but had no effect on MCF10A proliferation.

Vasectomy Affects Cysteine‐Rich Secretory Protein Expression Along the Human Epididymis and Its Association With Ejaculated Spermatozoa Following Vasectomy Surgical Reversal

The epididymis is essential for the acquisition of sperm fertilizing ability and forward motility. After vasectomy, the flux and composition of the epididymal fluid are modified, causing possible sequelae to the occluded excurrent duct. Some of these sequelae may not be reversible following vasovasostomy, affecting sperm physiology and their fertilizing ability. We previously demonstrated that the epididymal expression in men of a major glycoprotein secreted by the epididymis, cysteine-rich secretory protein 1 (CRISP1), and its encoding mRNA are affected by vasectomy. In this study we showed that following vasectomy, the increased level of CRISP1 is not due to a secretory defect but to its accumulation in the intraluminal compartment of the cauda epididymidis. Western blot analyses were performed to determine the amount of CRISP1 associated with spermatozoa of men who had undergone surgical vasectomy reversal. Spermatozoa of vasovasostomized men are characterized by a significant increase (P < .05) in CRISP1 levels when compared with normal donors. There was no linear correlation between CRISP1 levels and the period of time elapsed between vasectomy and vasovasostomy. CRISP1 was also present in seminal plasma of normal and vasovasostomized men, but not in vasectomized individuals. The soluble concentration of CRISP1 was significantly higher (P < .05) in seminal plasma of vasovasostomized men when compared with normal men. Knowing that one of the proposed functions of CRISP1 is to modulate sperm capacitation, we evaluated the level of tyrosine protein phosphorylation of 2 AXAP proteins of the fibrous sheath, p81 and p105. Spermatozoa of vasovasostomized men were characterized by a 50% increase of protein tyrosine phosphorylation when compared to spermatozoa of normal men (P < .05). Our results are discussed with regard to the fertilizing ability of ejaculated spermatozoa of some vasovasostomized men.

Microparticle and mitochondrial release during extended storage of different types of platelet concentrates

Abstract On activation, platelets release vesicles called microparticles (MPs). MPs are heterogeneous with regard to the presence or absence of mitochondria. We quantified MPs in platelet concentrates (PCs) taking their mitochondrial content into account. Platelet-rich plasma (PRP), buffy coat (BC) and apheresis (AP) PCs were tested through 7 days of storage. A combination of flow cytometry and spanning-tree progression analysis of density-normalized events (SPADE) was used to determine MP and mitochondrial release during storage. All the PC biochemical parameters complied with transfusion standards at all times. Platelet activation markers increased during storage and were higher for PRP than other types of PCs. Concentrations of MPs and extracellular mitochondria interpreted by SPADE algorithm were significantly higher in PRP than other in PCs and were stable throughout storage. The mode of preparation, rather than storage duration, impacts the release of MPs and mitochondria in PCs.

The Intracellular Localisation and Phosphorylation Profile of the Human 5-Lipoxygenase Δ13 Isoform Differs from That of Its Full Length Counterpart

5-Lipoxygenase (5-LO) catalyzes leukotriene (LT) biosynthesis by a mechanism that involves interactions with 5-lipoxygenase activating protein (FLAP) and coactosin-like protein (CLP). 5-LO splice variants were recently identified in human myeloid and lymphoid cells, including the catalytically inactive ∆13 isoform (5-LO∆13) whose transcript lacks exon 13. 5-LO∆13 inhibits 5-LO product biosynthesis when co-expressed with active full length 5-LO (5-LO1). The objective of this study was to investigate potential mechanisms by which 5-LO∆13 interferes with 5-LO product biosynthesis in transfected HEK293 cells. When co-expressed with 5-LO1, 5-LO∆13 inhibited LT but not 5-hydroxyeicosatetraenoic acid (5-HETE) biosynthesis. This inhibition was independent of 5-LO∆13—FLAP interactions since it occurred in cells expressing FLAP or not. In cell-free assays CLP enhances 5-LO activity through interactions with tryptophan-102 of 5-LO. In the current study, the requirement for W102 was extended to whole cells, as cells expressing the 5-LO1-W102A mutant produced little 5-LO products. W102A mutants of 5-LO∆13 inhibited 5-LO product biosynthesis as effectively as 5-LO∆13 suggesting that inhibition is independent of interactions with CLP. Confocal microscopy showed that 5-LO1 was primarily in the nucleoplasm whereas W102A mutants showed a diffuse cellular expression. Despite the retention of known nuclear localisation sequences, 5-LO∆13 was cytosolic and concentrated in ER-rich perinuclear regions where its effect on LT biosynthesis may occur. W102A mutants of 5-LO∆13 showed the same pattern. Consistent with subcellular distribution patterns, 5-LO∆13 was hyper-phosphorylated on S523 and S273 compared to 5-LO1. Together, these results reveal a role for W102 in nuclear targeting of 5-LO1 suggesting that interactions with CLP are required for nuclear localization of 5-LO1, and are an initial characterisation of the 5-LO∆13 isoform whose inhibition of LT biosynthesis appears independent of interactions with CLP and FLAP. Better knowledge of the regulation and properties of alternative 5-LO isoforms will contribute to understanding the complex regulation of LT biosynthesis.

On the cellular metabolism of the click chemistry probe 19-alkyne arachidonic acid

Alkyne and azide analogs of natural compounds that can be coupled to sensitive tags by click chemistry are powerful tools to study biological processes. Arachidonic acid (AA) is a FA precursor to biologically active compounds. 19-Alkyne-AA (AA-alk) is a sensitive clickable AA analog; however, its use as a surrogate to study AA metabolism requires further evaluation. In this study, AA-alk metabolism was compared with that of AA in human cells. Jurkat cell uptake of AA was 2-fold greater than that of AA-alk, but significantly more AA-Alk was elongated to 22:4. AA and AA-alk incorporation into and remodeling between phospholipid (PL) classes was identical indicating equivalent CoA-independent AA-PL remodeling. Platelets stimulated in the presence of AA-alk synthesized significantly less 12-lipoxygenase (12-LOX) and cyclooxygenase products than in the presence of AA. Ionophore-stimulated neutrophils produced significantly more 5-LOX products in the presence of AA-alk than AA. Neutrophils stimulated with only exogenous AA-alk produced significantly less 5-LOX products compared with AA, and leukotriene B4 (LTB4)-alk was 12-fold less potent at stimulating neutrophil migration than LTB4, collectively indicative of weaker leukotriene B4 receptor 1 agonist activity of LTB4-alk. Overall, these results suggest that the use of AA-alk as a surrogate for the study of AA metabolism should be carried out with caution.