While the mammalian macrophage phenotypes have been intensively studied in vitro, the dynamic of their phenotypic polarization has never been investigated in live vertebrates. We used the zebrafish as a live model to identify and trail macrophage subtypes. We generated a transgenic line whose macrophages expressing tumour necrosis factor alpha (tnfa), a key feature of classically activated (M1) macrophages, express fluorescent proteins Tg(mpeg1:mCherryF/tnfa:eGFP-F). Using 4D-confocal microscopy, we showed that both aseptic wounding and Escherichia coli inoculation triggered macrophage recruitment, some of which started to express tnfa. RT-qPCR on Fluorescence Activated Cell Sorting (FACS)-sorted tnfa+ and tnfa− macrophages showed that they, respectively, expressed M1 and alternatively activated (M2) mammalian markers. Fate tracing of tnfa+ macrophages during the time-course of inflammation demonstrated that pro-inflammatory macrophages converted into M2-like phenotype during the resolution step. Our results reveal the diversity and plasticity of zebrafish macrophage subsets and underline the similarities with mammalian macrophages proposing a new system to study macrophage functional dynamic.DOI: http://dx.doi.org/10.7554/eLife.07288.001

Objectives: Mesenchymal stem cells (MSCs) release extracellular vesicles (EVs) that display a therapeutic effect in inflammatory disease models. Although MSCs can prevent arthritis, the role of MSCs-derived EVs has never been reported in rheumatoid arthritis. This prompted us to compare the function of exosomes (Exos) and microparticles (MPs) isolated from MSCs and investigate their immunomodulatory function in arthritis.Methods: MSCs-derived Exos and MPs were isolated by differential ultracentrifugation. Immunosuppressive effects of MPs or Exos were investigated on T and B lymphocytes in vitro and in the Delayed-Type Hypersensitivity (DTH) and Collagen-Induced Arthritis (CIA) models.Results: Exos and MPs from MSCs inhibited T lymphocyte proliferation in a dose-dependent manner and decreased the percentage of CD4+ and CD8+ T cell subsets. Interestingly, Exos increased Treg cell populations while parental MSCs did not. Conversely, plasmablast differentiation was reduced to a similar extent by MSCs, Exos or MPs. IFN-γ priming of MSCs before vesicles isolation did not influence the immunomodulatory function of isolated Exos or MPs. In DTH, we observed a dose-dependent anti-inflammatory effect of MPs and Exos, while in the CIA model, Exos efficiently decreased clinical signs of inflammation. The beneficial effect of Exos was associated with fewer plasmablasts and more Breg-like cells in lymph nodes.Conclusions: Both MSCs-derived MPs and Exos exerted an anti-inflammatory role on T and B lymphocytes independently of MSCs priming. However, Exos were more efficient in suppressing inflammation in vivo. Our work is the first demonstration of the therapeutic potential of MSCs-derived EVs in inflammatory arthritis.

IntroductionMesenchymal stem cells (MSCs) are adult, multipotent, stem cells with immunomodulatory properties. The mechanisms involved in the capacity of MSCs to inhibit the proliferation of proinflammatory T lymphocytes, which appear responsible for causing autoimmune disease, have yet to be fully elucidated. One of the underlying mechanisms studied recently is the ability of MSCs to generate T regulatory (Treg) cells in vitro and in vivo from activated peripheral blood mononuclear cells (PBMC), T-CD4+ and also T-CD8+ cells. In the present work we investigated the capacity of MSCs to generate Treg cells using T-CD4+ cells induced to differentiate toward the proinflammatory Th1 and Th17 lineages.MethodsMSCs were obtained from mouse bone marrow and characterized according to their surface antigen expression and their multilineage differentiation potential. CD4+ T cells isolated from mouse spleens were induced to differentiate into Th1 or Th17 cells and co-cultured with MSCs added at day 0, 2 or 4 of the differentiation processes. After six days, CD25, Foxp3, IL-17 and IFN-γ expression was assessed by flow cytometry and helios and neuropilin 1 mRNA levels were assessed by RT-qPCR. For the functional assays, the ‘conditioned’ subpopulation generated in the presence of MSCs was cultured with concanavalin A-activated CD4+ T cells labeled with carboxyfluorescein succinimidyl ester. Finally, we used the encephalomyelitis autoimmune diseases (EAE) mouse model, in which mice were injected with MSCs at day 18 and 30 after immunization. At day 50, the mice were euthanized and draining lymph nodes were extracted for Th1, Th17 and Treg detection by flow cytometry.ResultsMSCs were able to suppress the proliferation, activation and differentiation of CD4+ T cells induced to differentiate into Th1 and Th17 cells. This substantial suppressive effect was associated with an increase of the percentage of functional induced CD4+CD25+Foxp3+ regulatory T cells and IL-10 secretion. However, using mature Th1 or Th17 cells our results demonstrated that while MSCs suppress the proliferation and phenotype of mature Th1 and Th17 cells they did not generate Treg cells. Finally, we showed that the beneficial effect observed following MSC injection in an EAE mouse model was associated with the suppression of Th17 cells and an increase in the percentage of CD4+CD25+Foxp3+ T lymphocytes when administrated at early stages of the disease.ConclusionsThis study demonstrated that MSCs contribute to the generation of an immunosuppressive environment via the inhibition of proinflammatory T cells and the induction of T cells with a regulatory phenotype. Together, these results might have important clinical implications for inflammatory and autoimmune diseases.