Programmed death-1 levels correlate with increased mortality, nosocomial infection and immune dysfunctions in septic shock patients

International audienceINTRODUCTION: Septic shock remains a major health care problem worldwide. Sepsis-induced immune alterations are thought to play a major role in patients' mortality and susceptibility to nosocomial infections. Programmed death-1 (PD-1) receptor system constitutes a newly described immunoregulatory pathway that negatively controls immune responses. It has recently been shown that PD-1 knock-out mice exhibited a lower mortality in response to experimental sepsis. The objective of the present study was to investigate PD-1-related molecule expressions in septic shock patients. METHODS: This prospective and observational study included 64 septic shock patients, 13 trauma patients and 49 healthy individuals. PD-1-related-molecule expressions were measured by flow cytometry on circulating leukocytes. Plasmatic interleukin (IL)-10 concentration as well as ex vivo mitogen-induced lymphocyte proliferation were assessed. RESULTS: We observed that septic shock patients displayed increased PD-1, PD-Ligand1 (PD-L1) and PD-L2 monocyte expressions and enhanced PD-1 and PD-L1 CD4+ T lymphocyte expressions at day 1-2 and 3-5 after the onset of shock in comparison with patients with trauma and healthy volunteers. Importantly, increased expressions were associated with increased occurrence of secondary nosocomial infections and mortality after septic shock as well as with decreased mitogen-induced lymphocyte proliferation and increased circulating IL-10 concentration. CONCLUSIONS: These findings indicate that PD-1-related molecules may constitute a novel immunoregulatory system involved in sepsis-induced immune alterations. Results should be confirmed in a larger cohort of patients. This may offer innovative therapeutic perspectives on the treatment of this hitherto deadly disease

Selective induction of cytokines in mouse brain infected with canine distemper virus: structural, cellular and temporal expression

International audienceWe have previously shown that, in experimentally inoculated mice, canine distemper virus (CDV), a neurotropic virus, selectively infects certain brain structures (hypothalamus, hippocampus, monoaminergic nuclei, etc). Here we demonstrate that tumor necrosis factor (TNF)-alpha, interleukin (IL)-1 beta and IL-6 transcripts are selectively expressed in these CDV-targeted structures, except in the dentate gyrus, where cytokines are induced without prior CDV replication. The time-course of TNF-alpha expression vs. viral replication in the hypothalamus was different from that in hippocampus. In addition, we show that a substantial number of neurons express TNF-alpha and IL-6. These findings provide new insights into the possible participation of cytokines in the neurological disorders triggered by CDV infection

Effect of pneumatic tube transport on T lymphocyte subsets analysis

International audienceBackground: Pneumatic tube system (PTS) for transportation of blood specimens may present with advantages for hospital organization as it provides faster tube transfer from medical wards to routine labs. These characteristics are expected to result in faster sample processing and decreased turnaround time, therefore benefiting the patient particularly in emergency units. However, PTS could affect sample quality and therefore laboratory results. Within the context of routine lab certification, effects of PTS on routine cellular immunology analyses, especially on determination of T lymphocyte subpopulations, need to be evaluated. Methods: Paired EDTA blood samples were collected from 30 healthy donors. For each pair, one sample was hand-delivered by a courier while the other was transported through a PTS of 2.4 km long (1.6 miles) with two 90-degree turns and one-U turn generating a speed of 5 m/s with a maximal acceleration of 2 g-force. The percentages of CD31 cells, CD41 and CD81 T-cells and their absolute counts were assessed by flow cytometry. Results: For every parameter, results measured by flow cytometry were not significantly different after transport by PTS or hand-delivery. Results comparison revealed an excellent linear correlation between both delivery methods (R ranged from 0.969 to 0.982; P < 0.01). Bland-Altman plots also showed good agreement, indicating that results were not influenced by the transport method. Conclusions: Our results suggest that, pending validation using clinical samples, PTS does not present with pre-analytical drawbacks and is thus a reliable system for blood samples transportation when performing T cell subset phenotyping.

Les astrocytes, cellules relais des interactions neuro-immunes dans le système nerveux central

L’astrocyte joue un rôle prépondérant dans la régulation des réponses immunes au sein du système nerveux central. Afin d’analyser les cascades moléculaires complexes suspectées de conduire à des atteintes neuroinflammatoires, nous avons établi in vitro un modèle d’interaction de lymphocytes T chroniquement activés avec des astrocytes humains (Dev-Ast), des astrocytes et oligodendrocytes de Rat en culture primaire, ou bien des précurseurs nerveux multipotents (lignée Dev). Nous montrons que les astrocytes humains et de Rat ont un profil réactif après contact avec les lymphocytes T activés. Leur sécrétion de cytokines pro-inflammatoires est élevée et se traduit par une augmentation de leur expression de métalloprotéases (MMP) et d’inhibiteurs endogènes spécifiques (TIMP). Ces modulations sont retrouvées dans les précurseurs nerveux. L’altération du système protéolytique MMP/TIMP est associée à une mort cellulaire induite par le contact lymphocytaire. Nous montrons que MMP et TIMP sont impliqués dans les cascades de signalisation de mort. Les lymphocytes T activés induisent une apoptose des précurseurs nerveux, médiée par Fas et leur sensibilité à l’apoptose est potentialisée par des inhibiteurs de MMP. Une stabilisation de Fas en surface par une inhibition de son clivage via des TIMP est suspectée. Les lymphocytes T activés induisent également l’apoptose d’oligodendrocytes immatures présents dans la culture gliale mixte. Cette mort est associée à une altération du système MMP/TIMP dans les astrocytes et est médiée par Fas dont l’expression est augmentée sur les oligodendrocytes. Ainsi, le dialogue établi entre lymphocytes T activés et cellules nerveuses induit une cascade moléculaire impliquant les cytokines pro-inflammatoires et le système protéolytique MMP/TIMP. Dans ce dialogue, le relais astro- cytaire apparaît comme crucial dans le processus qui conduit à la mort de cellules nerveuses. Une altération du système MMP/TIMP détectée chez des patients développant une atteinte neuroinflammatoire, la myélopathie associée à l’infection par le rétrovirus HTLV-1, renforce cette hypothèse

IL-7 Restores Lymphocyte Functions in Septic Patients

Abstract Septic syndrome is the leading cause of mortality for critically ill patients worldwide. Patients develop lymphocyte dysfunctions associated with increased risk of death and nosocomial infections. In this study, we performed preclinical experiments testing the potential of recombinant human IL-7 (rhIL-7) as a lymphostimulating therapy in sepsis. Circulating IL-7 and soluble IL-7 receptor α-chain (soluble CD127) concentrations were measured in plasma, whereas cellular CD127 expression was evaluated on circulating CD4+ and CD8+ lymphocytes from septic shock patients and healthy volunteers. Lymphocyte proliferation, IFN-γ production, STAT5 phosphorylation, and B cell lymphoma 2 induction were measured ex vivo in response to T cell stimulation in the presence or not of rhIL-7. We show that IL-7 pathway (plasmatic IL-7 concentration and cellular and soluble CD127 expressions) is not overtly altered and remains activable in septic patients. Most importantly ex vivo treatment of patients’ cells with rhIL-7 significantly improves lymphocyte functionality (CD4+ and CD8+ lymphocyte proliferations, IFN-γ production, STAT5 phosphorylation, and B cell lymphoma 2 induction after stimulation). To our knowledge, this constitutes the first report of rhIL-7 ability to restore normal lymphocyte functions in septic patients. These results support the rational for initiating a clinical trial testing rhIL-7 in septic shock

Assessment of a novel flow cytometry technique of one-step intracellular staining: Example of FOXP3 in clinical samples

International audienceBackground: By measuring multiple parameters on a single-cell basis, flow cytometry is a potent tool to dissect the phenotypes and functions of cell subsets. However, because this technique may be time-consuming, particularly for intracellular staining, it could be problematic for its use in daily routine or in large cohorts. Recently, a novel reagent has been developed to perform intracellular staining in one step. The objective of our study was thus to assess this new method in comparison with the reference technique by focusing on FOXP3 staining in clinical samples.Methods: Peripheral blood was collected from 15 HIV-1-infected patients, 5 critically ill patients, and 5 healthy volunteers and stained using the two different methods. Different subsets of FOXP3 positive cells were investigated by flow cytometry.Results: When comparing results obtained with the two techniques, no statistical differences between the percentages of CD4+FOXP3+, CD4+CD25+FOXP3+, and CD4+CD25+CD127-FOXP3+ cells were observed. Besides, a strong correlation between percentages of CD4+FOXP3+CD25+CD127- lymphocytes measured with both techniques was found in patients (r: 0.843, P < 0.001, intra-class correlation coefficient: 0.820, P < 0.001). Importantly, flow cytometry stainings obtained with the one-step method were very robust with an excellent intra-assay precision, a better discriminative power and correct stability and reproducibility of the staining even after blood storage.Conclusions: With a strong correlation between the percentages of FOXP3+ Tregs when compared with the reference method, a better staining quality, a shorter realization time and no need of isotype control, this one step procedure may represent an important improvement for a daily routine use of intracellular staining

IL-7 Restores Lymphocyte Functions in Septic Patients

International audienceAbstract Septic syndrome is the leading cause of mortality for critically ill patients worldwide. Patients develop lymphocyte dysfunctions associated with increased risk of death and nosocomial infections. In this study, we performed preclinical experiments testing the potential of recombinant human IL-7 (rhIL-7) as a lymphostimulating therapy in sepsis. Circulating IL-7 and soluble IL-7 receptor α-chain (soluble CD127) concentrations were measured in plasma, whereas cellular CD127 expression was evaluated on circulating CD4+ and CD8+ lymphocytes from septic shock patients and healthy volunteers. Lymphocyte proliferation, IFN-γ production, STAT5 phosphorylation, and B cell lymphoma 2 induction were measured ex vivo in response to T cell stimulation in the presence or not of rhIL-7. We show that IL-7 pathway (plasmatic IL-7 concentration and cellular and soluble CD127 expressions) is not overtly altered and remains activable in septic patients. Most importantly ex vivo treatment of patients’ cells with rhIL-7 significantly improves lymphocyte functionality (CD4+ and CD8+ lymphocyte proliferations, IFN-γ production, STAT5 phosphorylation, and B cell lymphoma 2 induction after stimulation). To our knowledge, this constitutes the first report of rhIL-7 ability to restore normal lymphocyte functions in septic patients. These results support the rational for initiating a clinical trial testing rhIL-7 in septic shock

Proof of concept study of mass cytometry in septic shock patients reveals novel immune alterations

Abstract Innovative single cell technologies such as mass cytometry (CyTOF) widen possibilities to deeply improve characterisation of immune alterations mechanisms in human diseases. So far, CyTOF has not been used in sepsis – a condition characterized by complex immune disorders. Here, we evaluated feasibility of CyTOF analysis in patients with septic shock. We designed a mass cytometry panel of 25 extracellular markers to study mononuclear cells from 5 septic shock patients and 5 healthy donors. We explored single-cell data with global and specific unsupervised approaches such as heatmaps, SPADE and viSNE. We first validated relevance of our CyTOF results by highlighting established immune hallmarks of sepsis, such as decreased monocyte HLA-DR expression and increased expressions of PD1 and PD-L1 on CD4 T cells and monocytes. We then showed that CyTOF analysis reveals novel aspects of sepsis-induced immune alterations, e.g. B cell shift towards plasma cell differentiation and uniform response of several monocyte markers defining an immune signature in septic patients. This proof of concept study demonstrates CyTOF suitability to analyse immune features of septic patients. Mass cytometry could thus represent a powerful tool to identify novel pathophysiological mechanisms and therapeutic targets for immunotherapy in septic shock patients