Influence of homocysteine on the physical structure and molecular mobility of elastin network in cultured arteries

The thermal and dielectric properties of the elastin network were investigated in arteries cultured with physiological and pathological concentrations of homocysteine, an aminoacid responsible of histological impairments in human arteries. The glass transition of this amorphous protein was investigated by Differential Scanning Calorimetry (DSC). To explore the molecular dynamics of the elastin network in the nanometer range, we used Thermally Stimulated Currents (TSC), a dielectric technique running at low frequency and measuring the dipolar reorientations in proteins subjected to a static electrical field. Combining TSC and DSC experiments with determination of the activation parameters of relaxation times reveals the molecular mobility of the proteins. The major differences in the relaxation behavior of elastin between arteries cultured with physiological and pathological concentrations of homocysteine are discussed

Changes in the physical structure and chain dynamics of elastin network in homocysteine-cultured arteries

The thermal and dielectric properties of the elastin network were investigated in arteries cultured with physiological and pathological concentrations of homocysteine, an aminoacid responsible of histological impairments in human arteries. The physical structure of this amorphous protein was investigated by differential scanning calorimetry (DSC). To explore the molecular dynamics of the elastin network in the nanometer range, we used thermally stimulated currents (TSC), a dielectric technique running at low frequency, and measuring the dipolar reorientations in proteins subjected to a static electrical field. Combining DSC and TSC experiments reveals the molecular mobility of the proteins, both in the glassy state and in the liquid state. Significant differences are evidenced in the physical structure and relaxation behavior of elastin network in cultured arteries (physiological and pathological concentrations of homocysteine) and discussed

Nod2 Mediates Susceptibility to Yersinia pseudotuberculosis in Mice

Nucleotide oligomerisation domain 2 (NOD2) is a component of the innate immunity known to be involved in the homeostasis of Peyer patches (PPs) in mice. However, little is known about its role during gut infection in vivo. Yersinia pseudotuberculosis is an enteropathogen causing gastroenteritis, adenolymphitis and septicaemia which is able to invade its host through PPs. We investigated the role of Nod2 during Y. pseudotuberculosis infection. Death was delayed in Nod2 deleted and Crohn's disease associated Nod2 mutated mice orogastrically inoculated with Y. pseudotuberculosis. In PPs, the local immune response was characterized by a higher KC level and a more intense infiltration by neutrophils and macrophages. The apoptotic and bacterial cell counts were decreased. Finally, Nod2 deleted mice had a lower systemic bacterial dissemination and less damage of the haematopoeitic organs. This resistance phenotype was lost in case of intraperitoneal infection. We concluded that Nod2 contributes to the susceptibility to Y. pseudotuberculosis in mice

A Key Role of Dendritic Cells in Probiotic Functionality

BACKGROUND: Disruption of the intestinal homeostasis and tolerance towards the resident microbiota is a major mechanism involved in the development of inflammatory bowel disease. While some bacteria are inducers of disease, others, known as probiotics, are able to reduce inflammation. Because dendritic cells (DCs) play a central role in regulating immune responses and in inducing tolerance, we investigated their role in the anti-inflammatory potential of probiotic lactic acid bacteria. METHODOLOGY/PRINCIPAL FINDINGS: Selected LAB strains, while efficiently taken up by DCs in vitro, induced a partial maturation of the cells. Transfer of probiotic-treated DCs conferred protection against 2, 4, 6-trinitrobenzenesulfonic acid (TNBS)-induced colitis. Protection was associated with a reduction of inflammatory scores and colonic expression of pro-inflammatory genes, while a high local expression of the immunoregulatory enzyme indolamine 2, 3 dioxgenase (IDO) was observed. The preventive effect of probiotic-pulsed DCs required not only MyD88-, TLR2- and NOD2-dependent signaling but also the induction of CD4+ CD25+ regulatory cells in an IL-10-independent pathway. CONCLUSIONS/SIGNIFICANCE: Altogether, these results suggest that selected probiotics can stimulate DC regulatory functions by targeting specific pattern-recognition receptors and pathways. The results not only emphasize the role of DCs in probiotic immune interactions, but indicate a possible role in immune-intervention therapy for IBD

Macrophage IL-1β-positive microvesicles exhibit thrombo-inflammatory properties and are detectable in patients with active juvenile idiopathic arthritis

ObjectiveIL-1β is a leaderless cytokine with poorly known secretory mechanisms that is barely detectable in serum of patients, including those with an IL-1β-mediated disease such as systemic juvenile idiopathic arthritis (sJIA). Leukocyte microvesicles (MVs) may be a mechanism of IL-1β secretion. The first objective of our study was to characterize IL-1β-positive MVs obtained from macrophage cell culture supernatants and to investigate their biological functions in vitro and in vivo. The second objective was to detect circulating IL-1β-positive MVs in JIA patients.MethodsMVs were purified by serial centrifugations from PBMCs, or THP-1 differentiated into macrophages, then stimulated with LPS ± ATP. MV content was analyzed for the presence of IL-1β, NLRP3 inflammasome, caspase-1, P2X7 receptor, and tissue factor (TF) using ELISA, Western blot, or flow cytometry. MV biological properties were studied in vitro by measuring VCAM-1, ICAM-1, and E-selectin expression after HUVEC co-culture and factor-Xa generation test was realized. In vivo, MVs’ ability to recruit leukocytes in a murine model of peritonitis was evaluated. Plasmatic IL-1β-positive MVs were studied ex vivo in 10 active JIA patients using flow cytometry.ResultsTHP-1-derived macrophages stimulated with LPS and ATP released MVs, which contained NLRP3, caspase-1, and the 33-kDa precursor and 17-kDa mature forms of IL-1β and bioactive TF. IL-1β-positive MVs expressed P2X7 receptor and released soluble IL-1β in response to ATP stimulation in vitro. In mice, MVs induced a leukocyte peritoneal infiltrate, which was reduced by treatment with the IL-1 receptor antagonist. Finally, IL-1β-positive MVs were detectable in plasma from 10 active JIA patients.ConclusionMVs shed from activated macrophages contain IL-1β, NLRP3 inflammasome components, and TF, and constitute thrombo-inflammatory vectors that can be detected in the plasma from active JIA patients

Microparticles from acute promyelocytic leukemia generate plasmin in a urokinase-dependent manner

International audienc

Microparticles from acute promyelocytic leukemia generate plasmin in a urokinase-dependent manner

International audienc

A new strategy to count and sort neutrophil‐derived extracellular vesicles: Validation in infectious disorders

International audienceNewly recognized polymorphonuclear neutrophil (PMNs) functions include the ability to release subcellular mediators such as neutrophil-derived extracellular vesicles (NDEVs) involved in immune and thrombo-inflammatory responses. Elevation of their plasmatic level has been reported in a variety of infectious and cardiovascular disorders, but the clinical use of this potential biomarker is hampered by methodological issues. Although flow cytometry (FCM) is currently used to detect NDEVs in the plasma of patients, an extensive characterization of NDEVs has never been done. Moreover, their detection remains challenging because of their small size and low antigen density. Therefore, the objective of the present study was first to establish a surface antigenic signature of NDEVs detectable by FCM and therefore to improve their detection in biological fluids by developing a strategy allowing to overcome their low fluorescent signal and reduce the background noise. By testing a large panel of 54 antibody specificities already reported to be positive on PMNs, we identified a profile of 15 membrane protein markers, including 4 (CD157, CD24, CD65 and CD66c) never described on NDEVs. Among them, CD15, CD66b and CD66c were identified as the most sensitive and specific markers to detect NDEVs by FCM. Using this antigenic signature, we developed a new strategy combining the three best antibodies in a cocktail and reducing the background noise by size exclusion chromatography (SEC). This strategy allowed a significant improvement in NDEVs enumeration in plasma from sepsis patients and made it feasible to efficiently sort NDEVs from COVID-19 patients. Altogether, this work opens the door to a more valuable measurement of NDEVs as a potential biomarker in clinical practice. A similar strategy could also be applied to improve detection by FCM of other rare subpopulations of EVs generated by tissues with limited access, such as vascular endothelium, cancer cells or placenta

Neutrophil extracellular traps are associated with the pathogenesis of diffuse alveolar hemorrhage in murine lupus

International audienceDiffuse alveolar hemorrhage (DAH) is a life-threatening complication of systemic lupus erythematosus (SLE) and systemic vasculitis. Although initially described to have antibacterial properties, increasing evidence suggests that neutrophil extracellular traps (NETs) have a detrimental role in both autoimmune diseases and acute lung injury. We investigated whether NETs could be detected in a murine model of pristane-induced lupus DAH and contribute to lung injury. Such NETs might constitute a therapeutic target. NETs were characterized by immunofluorescence staining of DNA, neutrophil elastase and citrullinated histones. Evaluation of lung injury was performed by haematoxylin-eosin staining and a quantification program. Clinical status of the mice was assessed by measurement of arterial oxygen saturation and survival curves after recombinant human deoxyribonuclease-1 (Rh-DNase-1) inhalations or polymorphonuclear neutrophil (PMN) depletion. Pristane was found to promote NETs formation in vitro and in vivo. Treatment of mice with Rh-DNase-1 inhalations cleared NETs and reduced lung injury. Clinical status improved significantly, with increased arterial oxygenation and survival. Following PMN depletion, NETs were absent with a subsequent reduction of lung injury and improved arterial oxygenation. These results support a pathogenic role of PMNs and NETs in lung injury during pristane-induced DAH. Targeting NETs with Rh-DNase-1 inhalations could constitute an interesting adjuvant therapy in human DAH

Figure 4

<p>Protective effect of intra-peritoneal administration of LAB-treated BMDCs on acute TNBS-induced colitis in BALB/c mice. Wallace inflammation scores were calculated after a TNBS challenge in mice either not treated (None) or intraperitoneally injected by untreated BMDCs (DC) or BMDCs treated with (A) Lr32 (Lr32-DC) or (B) Ls33 (Ls33-DC) strains. The comparison between the TNBS-control groups and the groups that received the corresponding untreated BMDCs (DC) was calculated using the Mann-Whitney U test (<0.01, **; p<0.001, ***). Percentage mortality and protection (reduction of the mean Wallace scores of mice treated with BMDCs, in relation to the mean score of TNBS control group) are also indicated. In A, the effect of Lr32-treated BMDCs was also compared to the intra-peritoneal administration of 10⁸ CFU of Lr32 bacteria (Lr32-IP) and in B, an additional group of mice was included, pre-treated with 3 intra-peritoneal administrations of prednisone (10 mg/kg), representing a clinically relevant standard treatment for Crohn's disease (36). Data represent the mean±SEM of two representative experiments (number of mice n = 10).</p