Platelet-derived extracellular vesicles in Huntington's disease.

The production and release of extracellular vesicles (EV) is a property shared by all eukaryotic cells and a phenomenon frequently exacerbated in pathological conditions. The protein cargo of EV, their cell type signature and availability in bodily fluids make them particularly appealing as biomarkers. We recently demonstrated that platelets, among all types of blood cells, contain the highest concentrations of the mutant huntingtin protein (mHtt)-the genetic product of Huntington's disease (HD), a neurodegenerative disorder which manifests in adulthood with a complex combination of motor, cognitive and psychiatric deficits. Herein, we used a cohort of 59 HD patients at all stages of the disease, including individuals in pre-manifest stages, and 54 healthy age- and sex-matched controls, to evaluate the potential of EV derived from platelets as a biomarker. We found that platelets of pre-manifest and manifest HD patients do not release more EV even if they are activated. Importantly, mHtt was not found within EV derived from platelets, despite them containing high levels of this protein. Correlation analyses also failed to reveal an association between the number of platelet-derived EV and the age of the patients, the number of CAG repeats, the Unified Huntington Disease Rating Scale total motor score, the Total Functional Capacity score or the Burden of Disease score. Our data would, therefore, suggest that EV derived from platelets with HD is not a valuable biomarker in HD.The study was funded by an operating grant from the Merck Sharpe & Dohme to F.C. who is also a recipient of a National Researcher career award from the Fonds de Recherche du Québec en santé (FRQS) providing salary support and operating funds. I.S.-A. was supported by a CIHR-Huntington Society of Canada postdoctoral fellowship. R.A.B. and S.L.M. are supported by a National Institute for Health Research (NIHR) award of a Biomedical Research Center to the University of Cambridge and Addenbrooke’s Hospital. E.B. is supported by the Canadian Institutes of Health Research. N.D. MD-MSc. also funded by CIHR and by Canadian Consortium on Neurodegeneration in Aging (CCNA). HLD and JPL hold a Desjardins scholarship from the Fondation du CHU de Québec. HLD hold a bourse d’excellence du Centre Thématique de Recherche en Neurosciences (CTRN) du CHU de Québec. The authors would like to thank all the students and staff who helped with the blood collections in Cambridge, Quebec City and Montreal and importantly, all patients and their families for being so generous with their time in participating to this stud

Revealing the diversity of extracellular vesicles using high-dimensional flow cytometry analyses

Extracellular vesicles (EV) are small membrane vesicles produced by cells upon activation and apoptosis. EVs are heterogeneous according to their origin, mode of release, membrane composition, organelle and biochemical content, and other factors. Whereas it is apparent that EVs are implicated in intercellular communication, they can also be used as biomarkers. Continuous improvements in pre-analytical parameters and flow cytometry permit more efficient assessment of EVs; however, methods to more objectively distinguish EVs from cells and background, and to interpret multiple single-EV parameters are lacking. We used spanning-tree progression analysis of density-normalized events (SPADE) as a computational approach for the organization of EV subpopulations released by platelets and erythrocytes. SPADE distinguished EVs, and logically organized EVs detected by high-sensitivity flow cytofluorometry based on size estimation, granularity, mitochondrial content, and phosphatidylserine and protein receptor surface expression. Plasma EVs were organized by hierarchy, permitting appreciation of their heterogeneity. Furthermore, SPADE was used to analyze EVs present in the synovial fluid of patients with inflammatory arthritis. Its algorithm efficiently revealed subtypes of arthritic patients based on EV heterogeneity patterns. Our study reveals that computational algorithms are useful for the analysis of high-dimensional single EV data, thereby facilitating comprehension of EV functions and biomarker development

Autoantibodies in Systemic Lupus Erythematosus Target Mitochondrial RNA

The mitochondrion supplies energy to the cell and regulates apoptosis. Unlike other mammalian organelles, mitochondria are formed by binary fission and cannot be directly produced by the cell. They contain numerous copies of a compact circular genome that encodes RNA molecules and proteins involved in mitochondrial oxidative phosphorylation. Whereas, mitochondrial DNA (mtDNA) activates the innate immune system if present in the cytosol or the extracellular milieu, it is also the target of circulating autoantibodies in systemic lupus erythematosus (SLE). However, it is not known whether mitochondrial RNA is also recognized by autoantibodies in SLE. In the present study, we evaluated the presence of autoantibodies targeting mitochondrial RNA (AmtRNA) in SLE. We quantified AmtRNA in an inducible model of murine SLE. The AmtRNA were also determined in SLE patients and healthy volunteers. AmtRNA titers were measured in both our induced model of murine SLE and in human SLE, and biostatistical analyses were performed to determine whether the presence and/or levels of AmtRNA were associated with clinical features expressed by SLE patients. Both IgG and IgM classes of AmtRNA were increased in SLE patients (n = 86) compared to healthy controls (n = 30) (p < 0.0001 and p = 0.0493, respectively). AmtRNA IgG levels correlated with anti-mtDNA-IgG titers (rs = 0.54, p < 0.0001) as well as with both IgG and IgM against β-2-glycoprotein I (anti-β2GPI; rs = 0.22, p = 0.05), and AmtRNA-IgG antibodies were present at higher levels when patients were positive for autoantibodies to double-stranded-genomic DNA (p < 0.0001). AmtRNA-IgG were able to specifically discriminate SLE patients from healthy controls, and were negatively associated with plaque formation (p = 0.04) and lupus nephritis (p = 0.03). Conversely, AmtRNA-IgM titers correlated with those of anti-β2GPI-IgM (rs = 0.48, p < 0.0001). AmtRNA-IgM were higher when patients were positive for anticardiolipin antibodies (aCL-IgG: p = 0.01; aCL-IgM: p = 0.002), but AmtRNA-IgM were not associated with any of the clinical manifestations assessed. These findings identify mtRNA as a novel mitochondrial antigen target in SLE, and support the concept that mitochondria may provide an important source of circulating autoantigens in SLE

Essentials of extracellular vesicles: posters on basic and clinical aspects of extracellular vesicles

The past decade has witnessed an exponential development in the field of extracellular vesicles. Sporadic observations have reached a critical level and the scientific community increasingly recognizes the potential biomedical significance of these subcellular structures present in all body fluids as significant components of the cellular secretome. The Educational Committee of the International Society for Extracellular Vesicles prepared two posters ("Basic aspects of extracellular vesicles" and "Clinical aspects of extracellular vesicles") to provide essential pieces of information on extracellular vesicles at glance for anyone not familiar with the field

Detection and Quantification of Microparticles from Different Cellular Lineages Using Flow Cytometry. Evaluation of the Impact of Secreted Phospholipase A2 on Microparticle Assessment

Microparticles, also called microvesicles, are submicron extracellular vesicles produced by plasma membrane budding and shedding recognized as key actors in numerous physio(patho)logical processes. Since they can be released by virtually any cell lineages and are retrieved in biological fluids, microparticles appear as potent biomarkers. However, the small dimensions of microparticles and soluble factors present in body fluids can considerably impede their quantification. Here, flow cytometry with improved methodology for microparticle resolution was used to detect microparticles of human and mouse species generated from platelets, red blood cells, endothelial cells, apoptotic thymocytes and cells from the male reproductive tract. A family of soluble proteins, the secreted phospholipases A2 (sPLA2), comprises enzymes concomitantly expressed with microparticles in biological fluids and that catalyze the hydrolysis of membrane phospholipids. As sPLA2 can hydrolyze phosphatidylserine, a phospholipid frequently used to assess microparticles, and might even clear microparticles, we further considered the impact of relevant sPLA2 enzymes, sPLA2 group IIA, V and X, on microparticle quantification. We observed that if enriched in fluids, certain sPLA2 enzymes impair the quantification of microparticles depending on the species studied, the source of microparticles and the means of detection employed (surface phosphatidylserine or protein antigen detection). This study provides analytical considerations for appropriate interpretation of microparticle cytofluorometric measurements in biological samples containing sPLA2 enzymes

Group IIA secreted phospholipase A2 controls skin carcinogenesis and psoriasis by shaping the gut microbiota

Besides promoting inflammation by mobilizing lipid mediators, group IIA secreted phospholipase A2 (sPLA2-IIA) prevents bacterial infection by degrading bacterial membranes. Here, we show that, despite the restricted intestinal expression of sPLA2-IIA in BALB/c mice, its genetic deletion leads to amelioration of cancer and exacerbation of psoriasis in distal skin. Intestinal expression of sPLA2-IIA is reduced after treatment with antibiotics or under germ-free conditions, suggesting its upregulation by gut microbiota. Metagenome, transcriptome, and metabolome analyses have revealed that sPLA2-IIA deficiency alters the gut microbiota, accompanied by notable changes in the intestinal expression of genes related to immunity and metabolism, as well as in the levels of various blood metabolites and fecal bacterial lipids, suggesting that sPLA2-IIA contributes to shaping of the gut microbiota. The skin phenotypes in Pla2g2a–/– mice are lost (a) when they are cohoused with littermate WT mice, resulting in the mixing of the microbiota between the genotypes, or (b) when they are housed in a more stringent pathogen-free facility, where Pla2g2a expression in WT mice is low and the gut microbial compositions in both genotypes are nearly identical. Thus, our results highlight a potentially new aspect of sPLA2-IIA as a modulator of gut microbiota, perturbation of which affects distal skin 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

Portrait of blood-derived extracellular vesicles in patients with Parkinson's disease.

The production of extracellular vesicles (EV) is a ubiquitous feature of eukaryotic cells but pathological events can affect their formation and constituents. We sought to characterize the nature, profile and protein signature of EV in the plasma of Parkinson's disease (PD) patients and how they correlate to clinical measures of the disease. EV were initially collected from cohorts of PD (n = 60; Controls, n = 37) and Huntington's disease (HD) patients (Pre-manifest, n = 11; manifest, n = 52; Controls, n = 55) - for comparative purposes in individuals with another chronic neurodegenerative condition - and exhaustively analyzed using flow cytometry, electron microscopy and proteomics. We then collected 42 samples from an additional independent cohort of PD patients to confirm our initial results. Through a series of iterative steps, we optimized an approach for defining the EV signature in PD. We found that the number of EV derived specifically from erythrocytes segregated with UPDRS scores corresponding to different disease stages. Proteomic analysis further revealed that there is a specific signature of proteins that could reliably differentiate control subjects from mild and moderate PD patients. Taken together, we have developed/identified an EV blood-based assay that has the potential to be used as a biomarker for PD

Bioactive lipids as biomarkers of adverse reactions associated with apheresis platelet concentrate transfusion

Platelet concentrate (PC) transfusion seeks to provide haemostasis in patients presenting severe central thrombocytopenia or severe bleeding. PCs may induce adverse reactions (AR) that can occasionally be severe (SAR). PCs contain active biomolecules such as cytokines and lipid mediators. The processing and storage of PCs creates so-called structural and biochemical storage lesions that accumulate when blood products reach their shelf life. We sought to investigate lipid mediators as bioactive molecules of interest during storage and review associations with adverse reactions post-transfusion. To facilitate understanding, we focused on single donor apheresis (SDA) PCs with approximately 31.8% of PCs being delivered in our setting. Indeed, pooled PCs are the most widely transfused products, but the study of a single donor lipid mediator is easier to interpret. We are investigating key lipid mediators involved in AR. Adverse reactions were closely monitored in accordance with current national and regional haemovigilance protocols. Residual PCs were analysed post-transfusion in a series of observations, both with and without severe reactions in recipients. A decrease in the lysophosphatidylcholine species to produce the lysophosphatidic acid species has been observed during storage and in the case of AR. Lysophosphatidic acid increased with primarily platelet-inhibitor lipids. Anti-inflammatory platelet-induced inhibition lipids were weakly expressed in cases of severe adverse reactions. We therefore propose that a decrease in lysophosphatidylcholine and an increase in lysophosphatidic acid can prospectively predict serious adverse transfusion reactions