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A strong bias related to age is observed in COVID-19 patients with pediatric subjects developing a milder disease than adults. We hypothesized that a specific SARS-CoV-2 effect conjugated with preexisting differences in the immune systems may explain this. Using flow cytometry, we investigated basal immune differences in a cohort consisting of 16 non-infected young and 16 aged individuals and further leveraged an in vitro whole blood model of SARS-CoV-2 infection so that functional differences could be mined as well. In short, blood diluted in culture media was incubated 5 or 24 h with the trimeric spike protein or controls. Following unsupervised analysis, we first confirmed that the immune lymphoid and myeloid systems in adults are less efficient and prone to develop higher inflammation than those in children. We notably identified in adults a higher CD43 lymphocyte expression, known for its potentially inhibitory role. The spike protein induced different responses between adults and children, notably a higher increase of inflammatory markers together with lower monocyte and B cell activation in adults. Interestingly, CD169, a CD43 ligand overexpressed in COVID-19 patients, was confirmed to be strongly modulated by the spike protein. In conclusion, the spike protein exacerbated the preexisting lower immune responsiveness and higher inflammatory potential in adults. Altogether, some of the markers identified may explain the marked age bias and be predictive of severity.
Abstract We assessed the expression of the cell adhesion molecule Sialoadhesin (CD169), a type I interferon-inducible receptor, on monocytes (mCD169) in 53 adult patients admitted to the hospital during the COVID-19 outbreak for a suspicion of SARS-CoV-2 infection. mCD169 was strongly overexpressed in 30 out of 32 (93.7%) confirmed COVID-19 cases, compared to three out of 21 (14.3%) patients for whom the diagnosis of COVID-19 was finally ruled out. mCD169 was associated with the plasma interferon alpha level and thrombocytopenia. mCD169 testing may be helpful for the rapid triage of suspected COVID-19 patients during an outbreak.
Vanin-1 is a membrane-anchored pantetheinase highly expressed in the gut and liver. It hydrolyzes pantetheine to pantothenic acid (vitamin B5) and the low-molecular-weight thiol cysteamine. The latter is believed to be a key regulating factor of several essential metabolic pathways, acting through sulfhydryl-disulfide exchange reactions between sulfhydryl groups of the enzymes and the oxidized form, cystamine. Its physiological importance remains to be elucidated, however. To explore this point, we developed Vanin-1-deficient mice that lack free cysteamine. We examined the susceptibility of deficient mice to intestinal inflammation, either acute (NSAID administration) or chronic (Schistosoma infection). We found that Vanin-1(-/-) mice better controlled inflammatory reaction and intestinal injury in both experiments. This protection was associated with increased gamma-glutamylcysteine synthetase activity and increased stores of reduced glutathione, as well as reduced inflammatory cell activation in inflamed tissues. Oral administration of cystamine reversed all aspects of the deficient phenotype. These findings suggest that one cysteamine function is to upregulate inflammation. Consequently, the pantetheinase activity of Vanin-1 molecule could be a target for a new anti-inflammatory strategy.
Decreased expression of human leukocyte antigen-DR on monocytes (mHLA-DR) is recognized as the most appropriate marker for the monitoring of immune alterations in septic patients and critically ill subjects. Its measurement has been established for years by flow cytometry, but remains under-used due to pre-analytical constraints. The objectives of the present work were to develop a rapid and robust one-step protocol.A novel, simplified protocol has been developed to measure mHLA-DR in whole blood using flow cytometry. It is a one-step procedure that includes red cell lysis, antibodies, and fixative reagents. It has been compared to the standardized routine protocol in two consecutive cohorts of septic shock patients (n = 37). Finally, the protocol was applied to a few subjects in point-of-care settings, by collecting capillary blood from fingerpricks.Strong correlation was observed between the one-step method and routine protocol in 24 patients. After testing several stabilizing agents, the procedure was further optimized by adding a low-dose formaldehyde to the stain and lyse solution. This improved method was tested in a second cohort of 13 patients, and again strongly correlated to the routine protocol. Finally, the fingerprick and venous puncture samples were shown to provide similar results.The present work demonstrates the feasibility of a bedside protocol for flow cytometry measurement of mHLA-DR in critically ill subjects. This helps overcome pre-analytical constraints previously identified, which have limited wider use of this biomarker in intensive care units. In addition, preliminary results from fingerprick samples are promising.
Les lymphocytes t sont une composante essentielle du systeme immunitaire, ils sont issus de precurseurs loges dans la moelle osseuse qui doivent subir une maturation prealable dans le thymus. Cette migration de la moelle osseuse vers le thymus s'effectue via le sang, et les mecanismes impliques sont encore mal connus, au meme titre que les caracteristiques des cellules qui accomplissent ce transfert. Le thymus est un organe complexe qui assure une expansion massive des precurseurs et leur stricte selection, de sorte que les thymocytes qui quittent le thymus, et deviennent des lymphocytes t, sont aptes a repondre a une agression de l'organisme. Les etapes de proliferation et de mort cellulaire sont accompagnees de changements phenotypiques notables des thymocytes, cette dynamique s'ajoute a la rarete des precurseurs issus de la moelle osseuse et rend l'etude de la colonisation thymique difficile. Vanin-1 est une nouvelle glycoproteine decrite en 96 dans le laboratoire, qui a ete proposee pour participer a cette etape de migration, et dont la distribution perivasculaire suggerait que les pericytes controlent la colonisation thymique au meme titre que l'endothelium. Cette theorie nous a conduit a rechercher d'autres structures vasculaires ou perivasculaires, et a etudier leur role dans la colonisation thymique. En parallele, l'etude de vanin-1 et de la famille des genes vanin chez l'homme et la souris s'est poursuivie. Nous avons montre que la reactivite perivasculaire de l'anticorps utilise dans les etudes precedentes ne correspond pas a vanin-1, et que vanin-1 n'est exprimee dans le thymus que par de rares cellules medullaires. Le role de vanin-1 dans la colonisation thymique est remis en question, et sa fonction principale se situe au niveau de la reconstitution thymique par les thymocytes radioresistants, apres irradiation. Nous avons realise l'inactivation genique de vanin-1 chez la souris, et le phenotype observe est en accord avec ces conclusions. La distribution de vanin-1 dans le reste de l'organisme, ses caracteristiques structurales, sa place dans une famille multigenique, et le phenotype des souris vanin-1 -/- nous orientent vers un role de vanin-1 dans le systeme neuro-endocrinien du controle de la reponse au stress.
Congenital macrothrombocytopenia is a family of rare diseases, of which a significant fraction remains to be genetically characterized. To analyze cases of unexplained thrombocytopenia, 27 individuals from a patient cohort of the Bleeding and Thrombosis Exploration Center of the University Hospital of Marseille were recruited for a high-throughput gene sequencing study. This strategy led to the identification of two novel FLI1 variants (c.1010G>A and c.1033A>G) responsible for macrothrombocytopenia. The FLI1 variant carriers' platelets exhibited a defect in aggregation induced by low-dose adenosine diphosphate (ADP), collagen and thrombin receptor-activating peptide (TRAP), a defect in adenosine triphosphate (ATP) secretion, a reduced mepacrine uptake and release and a reduced CD63 expression upon TRAP stimulation. Precise ultrastructural analysis of platelet content was performed using transmission electron microscopy and focused ion beam scanning electron microscopy. Remarkably, dense granules were nearly absent in the carriers' platelets, presumably due to a biogenesis defect. Additionally, 25–29% of the platelets displayed giant α-granules, while a smaller proportion displayed vacuoles (7–9%) and autophagosome-like structures (0–3%). In vitro study of megakaryocytes derived from circulating CD34+ cells of the carriers revealed a maturation defect and reduced proplatelet formation potential. The study of the FLI1 variants revealed a significant reduction in protein nuclear accumulation and transcriptional activity properties. Intraplatelet flow cytometry efficiently detected the biomarker MYH10 in FLI1 variant carriers. Overall, this study provides new insights into the phenotype, pathophysiology and diagnosis of FLI1 variant-associated thrombocytopenia.
Abstract Introduction: Phospho-epitopes are difficult to detect by flow cytometry, currently requiring dedicated techniques and buffers, a total work time of 2 to 4 hours, 37°C and ice incubations, and 4 to 8 wash steps. The reference methods also uses methanol which is toxic for the user and harmful for the other cell markers, what compromises some multi-parametric studies. The FoxP3 marker is incompatible with “harsh” P-epitopes detection methods, rendering even more challenging the studies of Tregs functions. Methods: Here, we used a new commercially available kit; named PerFix EXPOSE (Phospho Epitopes Exposure Kit; for Research Use Only) based on multiple innovations in the permeabilization, staining, and wash steps. It is devoid of methanol and supports staining of all common P-epitopes with one single procedure of about 1 hour. Since many extra-cellular markers can be combined together with the anti-phospho markers, we evaluated also various FoxP3 clones and conjugates, and tried some procedure optimisations. The best conditions were then used to analyse the ex-vivo functionality of FoxP3+ cells: The IL-2 signalling pathway especially. Results: PerFix EXPOSE allows for the simultaneous detection of some antigens that are otherwise incompatible, such as FOXP3 and p-STATs. An IL-2 dose-response curve could be easily generated directly from fresh whole blood that confirmed on normal donors a 10 to 100-fold difference in sensitivity to IL-2 between Tregs and other T cells.
An unbiased approach to SARS-CoV-2-induced immune dysregulation has not been undertaken so far. We aimed to identify previously unreported immune markers able to discriminate COVID-19 patients from healthy controls and to predict mild and severe disease.An observational, prospective, multicentric study was conducted in patients with confirmed mild/moderate (n = 7) and severe (n = 19) COVID-19. Immunophenotyping of whole-blood leukocytes was performed in patients upon hospital ward or intensive care unit admission and in healthy controls (n = 25). Clinically relevant associations were identified through unsupervised analysis.Granulocytic (neutrophil, eosinophil, and basophil) markers were enriched during COVID-19 and discriminated between patients with mild and severe disease. Increased counts of CD15+CD16+ neutrophils, decreased granulocytic expression of integrin CD11b, and Th2-related CRTH2 downregulation in eosinophils and basophils established a COVID-19 signature. Severity was associated with emergence of PD-L1 checkpoint expression in basophils and eosinophils. This granulocytic signature was accompanied by monocyte and lymphocyte immunoparalysis. Correlation with validated clinical scores supported pathophysiological relevance.Phenotypic markers of circulating granulocytes are strong discriminators between infected and uninfected individuals as well as between severity stages. COVID-19 alters the frequency and functional phenotypes of granulocyte subsets with emergence of CRTH2 as a disease biomarker.
