Microglia and dendritic cells as a source of IL-6 in a mouse model of multiple sclerosis

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Abstract

Multiple sclerosis (MS) is a complex autoimmune disease of the central nervous system (CNS), characterized by myelin sheath destruction and compromised nerve signal transmission. Understanding the molecular mechanisms driving MS development is critical due to its early onset, chronic course, and therapeutic approaches based only on symptomatic treatment. Cytokines are known to play a pivotal role in the pathogenesis of MS, with interleukin-6 (IL-6) being one of the key mediators. This study investigates the contribution of IL-6 produced by microglia and dendritic cells to the development of experimental autoimmune encephalomyelitis (EAE), a widely used mouse model of MS. Mice with conditional inactivation of IL-6 in CX3CR1+ cells, including microglia, or CD11c+ dendritic cells, displayed less severe symptoms as compared to their wild-type counterparts. Mice with microglial IL-6 deletion exhibited an elevated proportion of regulatory T cells and a reduced percentage of pathogenic IFNγ-producing CD4+ T cells, accompanied by a decrease in pro-inflammatory monocytes, in the CNS at the peak of EAE. At the same time, deletion of IL-6 from microglia resulted in an increase of CCR6+ T cells and GM-CSF-producing T cells. Conversely, mice with IL-6 deficiency in dendritic cells showed not only the previously described increase in the proportion of regulatory T cells and a decrease in the proportion of TH17 cells, but also a reduction in the production of GM-CSF and IFNγ in secondary lymphoid organs. In summary, IL-6 functions during EAE depend on both the source and the localization of the immune response: microglial IL-6 exerts both pathogenic and protective functions specifically in the CNS, whereas dendritic cell-derived IL-6, in addition to being critically involved in the balance of regulatory T cells and TH17 cells, may stimulate the production of cytokines associated with the pathogenetic functions of T cells.

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V. S. Gogoleva

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Author for correspondence.
Email: violettegogoleva@mail.ru

Center for Precision Genome Editing and Genetic Technologies for Biomedicine

Russian Federation, 117997, Moscow

Q. Chi Nguyen

Lomonosov Moscow State University

Email: violettegogoleva@mail.ru

Faculty of Biology

Russian Federation, 119991, Moscow

M. S. Drutskaya

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: violettegogoleva@mail.ru

Center for Precision Genome Editing and Genetic Technologies for Biomedicine

Russian Federation, 117997, Moscow

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2. Fig. 1. Mice with tamoxifen-dependent IL-6 inactivation in CX3CR1+ microglia are resistant to the development of EAE. a is the scheme of the experiment. Tamoxifen was administered to mice at a rate of 75 mcg/g for 5 days. Further, 7-14 days after the tamoxifen course, IL-6 removal was detected in both tissue-resident macrophages and monocytes, but after 28 days the pool of monocytes from the bone marrow was updated and IL-6 deficiency was observed only in tissue-resident macrophages, including microglia; b – concentration of IL-6 in the supernatant of sorted CX3CR1+ monocytes isolated from the peripheral blood of Cx3cr1CreER × Il6fl/fl mice on 0, 14, 28 days after tamoxifen administration and activated LPS for 4 hours; ND – not detected; c – concentration of IL-6 in the supernatant of sorted CX3CR1+ microglia isolated from the central nervous system of mice Cx3cr1CreER × Il6fl/fl on day 28 after administration of tamoxifen and activated LPS for 4 hours; d – dynamics of the development of clinical symptoms of EAE in wild-type mice (Il6fl/fl), mice with IL-6 removal only in microglia (Il6ΔMG) and mice with IL–6 removal only in dendritic cells (Il6ΔDC) immunized with MOG35-55 peptide in a complete Freund adjuvant. The results are presented as an average value of ± SEM and confirmed in three independent experiments with a minimum number of mice in the group n = 4 in each experiment

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3. Fig. 2. IL-6 produced by microglia stimulates the development of pathogenetic monocytes in the central nervous system and the production of IFNy at the peak of EAE. a – The proportion of LY6HIMHCII+ cells from CD45+CD11b+ cells in the central nervous system at the peak of EAE; b - the absolute number of LY6HIMHCII+ cells in the central nervous system at the peak of EAE; c - the proportion of IFNy–producing cells restimulated by PMA/ionomycin from CD4+TCRß+ cells in the central nervous system at the peak of EAE; d – the proportion of FoxP3+ Treg from CD4+TCRß+ cells in the central nervous system at the peak of EAE. Combined (a, c and d) or representative data (b) with the number of mice of each genotype n = 5-6 are presented. The data are presented as an average ± SEM. The Student's t-test or the Mann–Whitney test were used; * p < 0.05; ** p < 0.01

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4. 3. Deletion of the gene encoding IL-6 in microglia leads to increased migration of T cells and induction of GM-CSF-producing CD4+ T cells in the central nervous system. a is the percentage of CCR6+ cells from CD4+TCRß+ cells in the central nervous system at the peak of EAE. b is the proportion of GM-CSF-producing cells restimulated by PMA/ionomycin from CD4+TCRß+ cells in the central nervous system at the peak of EAE. Combined (a) or representative data (b) with the number of mice of each genotype n = 5-6 are presented. The data are presented as an average ± SEM. The Student's t-test or the Mann–Whitney test were used; * p < 0.05; *** p < 0.001; ns – unreliable differences

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5. 4. IL-6 from dendritic cells stimulates the production of cytokines by CD4+ T cells in peripheral lymphoid organs at the peak of EAE. a – Representative dot diagrams (left) and the percentage (right) of RORyt+ and FoxP3+ CD4+ T cells isolated from the lymph nodes of Il6fl/fl and Il6ΔDC mice at the peak of EAE. b–d is the percentage of IL-17A-, GM-CSF-, IFNy-producing CD4+ T cells isolated from the spleen and lymph nodes and restimulated with PMA/ionomycin. The results are presented as an average value of ± SEM and confirmed in two independent experiments. The Student's t-test or the Mann–Whitney test were used; * p < 0.05; *** p < 0.001; ns – unreliable differences

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