Identifying factors that shape IL-15 expression on antigen presenting cells in the context of multiple sclerosis

L’IL-15 est une cytokine pléiotropique qui joue un rôle important dans la prolifération, l’activation, et la survie de plusieurs types cellulaires, dont les lymphocytes T CD8. Nous avions précédemment montré que l’expression de l’IL-15 est augmentée dans les cerveaux de personnes atteintes de la sclérose en plaques (SP) par rapport aux cerveaux témoins. De plus, un pourcentage plus élevé des cellules immunitaires provenant du sang des patients atteints de la SP, dont les monocytes, expriment l’IL-15 par rapport aux cellules des donneurs témoins. Nous avions aussi prouvé que l’expression de cette cytokine peut être induite en stimulant des monocytes avec des facteurs pro-inflammatoires, tels que l’IFNγ et le LPS. Cependant, les facteurs qui contribuent à la forte expression de l’IL-15 dans les cerveaux des patients atteints de la SP demeurent inconnus. Finalement, la contribution relative des macrophages et des microglies dans le cerveau à cette forte expression n’est pas encore connue. Notre hypothèse est que des facteurs pro-inflammatoires ou des signaux de danger exprimés dans le cerveau pendant la pathobiologie de la SP contribuent à l’augmentation des niveaux d’IL-15 sur les cellules myéloïdes. Notre objectif est d’identifier ces facteurs et de déterminer les voies de signalisation impliquées dans ce mécanisme. Nous avons identifié le GM-CSF, une cytokine impliquée dans la pathologie de la SP et de ses modèles murins, comme facteur inducteur de l’IL-15. En effet, des monocytes de donneurs sains et de patients SP différenciés en macrophages non-polarisés, ou polarisés vers un phénotype M1 ou M2, ont augmenté significativement l’expression de l’IL-15 en pourcentage de cellules positives ou en niveau par cellule, suite à une exposition au GM-CSF. De plus, le GM-CSF a augmenté le niveau de l’IL-15 par cellule sur les microglies primaires, adultes, humaines, mais il n’a pas modifié le pourcentage des microglies exprimant l’IL-15. Le GM-CSF a aussi augmenté l’expression du CD80 et de l’ICAM-1 sur les macrophages, et il a induit une plus forte sécrétion de l’IL-6, l’IL-27, et du CXCL10 par ces cellules myéloïdes. Étonnamment, le GM-CSF a diminué la proportion des monocytes ex vivo qui exprime l’IL-15. Cependant, le GM-CSF a déclenché majoritairement les mêmes voies de signalisation dans les monocytes et les macrophages, dont les voies STAT3, STAT5, et ERK1/2. En utilisant des inhibiteurs de ces voies, nous avons pu conclure que l’ERK1/2 est impliqué dans ce mécanisme dans ces deux populations. Finalement, les macrophages exposés au GM-CSF ont démontré une capacité plus élevée d’induire la production des cytokines et des produits cytotoxiques par des lymphocytes T CD8 dans un système de coculture autologue, par rapport aux macrophages non-traités. Même si l’IL-15 est suffisant pour stimuler les lymphocytes T CD8, nos expériences de blocage de l’IL-15 ont démontré que cette cytokine n’est pas nécessaire dans ce système de coculture. Nous pensons que le GM-CSF augmente l’expression et la sécrétion de nombreux facteurs qui ensemble contribuent à une plus forte activation des lymphocytes CD8.IL-15 is a pleiotropic cytokine with an important role in the activation, proliferation, and survival of immune cell populations including CD8 T cells. We and others have shown elevated IL-15 levels in MS brain tissue compared to non-MS controls. Additionally, a greater percentage of immune cells, including monocytes, from the peripheral blood of MS patients express IL-15 compared to cells from healthy donors. We have also shown that IL-15 can be induced on monocytes with pro-inflammatory stimuli, including IFNγ and LPS. However, it remains unclear what causes elevated expression of IL-15 on differentiated macrophages in the brain. Additionally, the relative contribution of peripherally-derived macrophages and CNS-resident microglia to elevated IL-15 expression in the MS brain is not known. We hypothesize that inflammatory or danger signals present in the brain of MS patients trigger IL-15 expression by myeloid cells. We aim to identify these triggers and describe the signaling pathways implicated in this mechanism. We found that GM-CSF, a cytokine that is increasingly being recognized as integral to MS and its animal models, significantly increased the proportion of IL-15-expressing or IL-15 levels on a per cell basis on unpolarized and M1- and M2- polarized monocyte-derived macrophages from healthy donors and MS donors. GM-CSF enhanced IL-15 levels on primary, human adult microglia, but did not increase the percentage of IL-15 expressing cells. GM-CSF also increased the expression of CD80 and ICAM-1 on macrophages, and enhanced the secretion of IL-6, IL-27, and CXCL10. Interestingly, GM-CSF decreased the percentage of IL-15+ ex vivo monocytes; we can therefore conclude that GM-CSF has opposing effects on IL-15 expression on monocytes and differentiated macrophages/ microglia. Nevertheless, we found that GM-CSF triggered mostly the same signaling pathways in human monocytes and macrophages, the most significant being STAT3, STAT5, and ERK1/2. By employing pharmacological inhibitors of these pathways, we found that ERK1/2 is implicated in the GM-CSF-mediated effect on IL-15 expression in both populations. Lastly, GM-CSF treated macrophages had a greater capacity to promote CD8 T cell cytotoxicity and cytokine production than untreated macrophages in an autologous co-culture system. Although IL-15 was sufficient for this effect, we found by blocking IL-15 signaling that it was not necessary. We posit that GM-CSF upregulates the expression and secretion of many pro-inflammatory factors by macrophages that together enhance CD8 T cell activation

The IL‐27/IL‐27R axis is altered in CD4 + and CD8 + T lymphocytes from multiple sclerosis patients

International audienceObjectives: Pro- and anti-inflammatory properties have been attributed to interleukin-27 (IL-27). Nevertheless, the impact of this cytokine on chronic inflammatory diseases such as multiple sclerosis (MS) remains ill-defined. We investigated the biology of IL-27 and its specific receptor IL-27Rα in MS patients.Methods: Levels of IL-27 and its natural antagonist (IL-27-Rα) were measured by ELISA in biological fluids. CD4+ and CD8+ T lymphocytes were isolated from untreated relapsing-remitting MS patients and healthy donors. Transcriptome-wide analysis compared T-cell subsets stimulated or not with IL-27. Expression of the IL-27Rα, key immune factors, STAT phosphorylation and cytokine production was assessed by flow cytometry.Results: We observed elevated levels of IL-27 in the serum and cerebrospinal fluid of MS patients compared with controls. Moreover, we show that specific IL-27-mediated effects on T lymphocytes are reduced in MS patients including the induction of PD-L1. IL-27-triggered STAT3 signalling pathway is enhanced in CD4+ and CD8+ T lymphocytes from MS patients. Elevated IL-27Rα levels in serum from MS patients are sufficient to impair the capacity of IL-27 to act on immune cells. We demonstrate that shedding of IL-27Rα by activated CD4+ T lymphocytes from MS patients contributes to the increased IL-27Rα peripheral levels and consequently can dampen the IL-27 responsiveness.Conclusion: Our work identifies several mechanisms that are altered in the IL-27/IL-27R axis in MS patients, especially in T lymphocytes. Our results underline the importance of characterising the biology of cytokines in human patients prior to design new therapeutics

Identification of SARS-CoV-2-specific immune alterations in acutely ill patients

Dysregulated immune profiles have been described in symptomatic patients infected with SARS-CoV-2. Whether the reported immune alterations are specific to SARS-CoV-2 infection or also triggered by other acute illnesses remains unclear. We performed flow cytometry analysis on fresh peripheral blood from a consecutive cohort of (a) patients hospitalized with acute SARS-CoV-2 infection, (b) patients of comparable age and sex hospitalized for another acute disease (SARS-CoV-2 negative), and (c) healthy controls. Using both data-driven and hypothesis-driven analyses, we found several dysregulations in immune cell subsets (e.g., decreased proportion of T cells) that were similarly associated with acute SARS-CoV-2 infection and non-COVID-19-related acute illnesses. In contrast, we identified specific differences in myeloid and lymphocyte subsets that were associated with SARS-CoV-2 status (e.g., elevated proportion of ICAM-1+ mature/activated neutrophils, ALCAM+ monocytes, and CD38+CD8+ T cells). A subset of SARS-CoV-2-specific immune alterations correlated with disease severity, disease outcome at 30 days, and mortality. Our data provide an understanding of the immune dysregulation specifically associated with SARS-CoV-2 infection among acute care hospitalized patients. Our study lays the foundation for the development of specific biomarkers to stratify SARS-CoV-2-positive patients at risk of unfavorable outcomes and to uncover candidate molecules to investigate from a therapeutic perspective