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. 2016 Oct;46(10):2454-2466.
doi: 10.1002/eji.201546274.

Selective depletion of CD11c+ CD11b+ dendritic cells partially abrogates tolerogenic effects of intravenous MOG in murine EAE

Limei Wang  1   2 , Zichen Li  1 , Bogoljub Ciric  1 , Farinaz Safavi  1 , Guang-Xian Zhang  1 , Abdolmohamad Rostami  3
Affiliations

Selective depletion of CD11c+ CD11b+ dendritic cells partially abrogates tolerogenic effects of intravenous MOG in murine EAE

Limei Wang et al. Eur J Immunol. 2016 Oct.

Abstract

Intravenous (i.v.) injection of a soluble myelin antigen can induce tolerance, which effectively ameliorates experimental autoimmune encephalomyelitis (EAE). We have previously shown that i.v. myelin oligodendrocyte glycoprotein (MOG) induces tolerance in EAE and expands a subpopulation of tolerogenic CD11c+ CD11b+ dendritic cells (DCs) with an immature phenotype having low expression of IA and co-stimulatory molecules CD40, CD86, and CD80. Here, we further investigate the role of tolerogenic DCs in i.v. tolerance by injecting clodronate-loaded liposomes, which selectively deplete CD11c+ CD11b+ and immature DCs, but not CD11c+ CD8+ DCs and mature DCs. I.v. MOG-induced suppression of EAE was partially, yet significantly, blocked by CD11c+ CD11b+ DC depletion. While i.v. MOG inhibited IA, CD40, CD80, CD86 expression and induced TGF-β, IL-27, IL-10 production in CD11c+ CD11b+ DCs, these effects were abrogated after injection of clodronate-loaded liposomes. Depletion of CD11c+ CD11b+ DCs also precluded i.v. autoantigen-induced T-cell tolerance, such as decreased production of IL-2, IFN-γ, IL-17 and numbers of IL-2+ , IFN-γ+ , and IL-17+ CD4+ T cells, as well as an increased proportion of CD4+ CD25+ Foxp3+ regulatory T cells and CD4+ IL-10+ Foxp3- Tr1 cells. CD11c+ CD11b+ DCs, through low expression of IA and costimulatory molecules as well as high expression of TGF-β, IL-27, and IL-10, play an important role in i.v. tolerance-induced EAE suppression.

Keywords: Dendritic cell; Experimental autoimmune encephalomyelitis; Immune tolerance; Multiple sclerosis.

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Conflict of interest statement

Disclosure of potential conflict of interest

The authors declare no financial or commercial conflict of interest.

Figures

Fig. 1
Fig. 1. Clodronate-loaded liposomes selectively deplete CD11c+CD11b+ DCs or iDCs
Naïve C57BL/6 mice were i.p. injected with PBS-loaded or clodronate-loaded liposomes, splenocytes were harvested at 24 hours later and analyzed with flow cytometry. (A) Percentages of DCs (CD11c+) among splenocytes (left). Results were statistically analyzed and shown as mean ± SEM (n=3 each group) (right). (B) CD11c+ cells were then gated and percentages of CD11b+ and CD8+ cells were analyzed (left). Results were statistically analyzed and shown as mean ± SEM (n=3 each group) (right). (C) Expression of CD11c vs. CD83, CD80, and CD86 in splenocytes (left). Results were statistically analyzed and shown as mean ± SEM (n=3 each group) (right). (D) Expression of CD83, CD80, and CD86 in total CD11c+ cells and in CD11b+, CD8+, CD11b−CD8− cells of gated CD11c+ cells. *p<0.05, ***p<0.001, unpaired Student’s t test. One representative of 3 experiments is shown.
Fig. 2
Fig. 2. Clodronate-loaded liposome partly block i.v. MOG-induced EAE suppression
Mice were immunized with MOG35–55 in CFA to induce EAE. MOG35–55 or PBS was i.v. injected at days 0, 3, and 6 p.i. Clodronate- or PBS-loaded liposome was i.p. injected 24 hours before and 14 days p.i. (A) Clinical signs were scored daily following a 0–5 scale. Data were pooled from three independent experiments and presented as mean clinical score ± SEM (n=11–15 each group). (B) On day 21 p.i., spinal cords were harvested and 5-µm sections were stained with H&E and LFB. Demyelination areas are designated by arrows. (Magnifications, × 10). (C) Inflammation and demyelination were scored using a 0–3 scale and expressed as mean scores ± SEM (n=3 each group). *p<0.05, ***p<0.001, two-way ANOVA test. One representative of 3 experiments is shown.
Fig. 3
Fig. 3. I.v. tolerance induced increase of CD11c+CD11b+ DCs is abrogated by injection of clodronate-loaded liposomes
Mice immunized for EAE induction were i.v. injected with MOG or PBS, while PBS-loaded or clodronate-loaded liposomes were i.p. injected. Splenocytes were isolated 21 days p.i. and analyzed by flow cytometry. (A) FACS profile of macrophages (F4/80+ cells) in total splenocytes (left). Results were statistically analyzed and shown as mean ± SEM (n=3 each group) (right). (B) M1 (IA+) and M2 (CD206+) macrophages in gated F4/80+ cells (left). Results were statistically analyzed and shown as mean ± SEM (n=3 each group) (right). (C) DCs (CD11c+ cells) in total splenocytes (left). Results were statistically analyzed and shown as mean ± SEM (n=3 each group) (right). (D) CD11c+CD11b+ DCs (CD11b+ cells in gated CD11c+ cells) and CD11c+CD8+ DCs (CD8+ cells in gated CD11c+ cells) (left). Results were statistically analyzed and data shown as mean ± SEM (n=3 each group). *p<0.05, ** p<0.01, ***p<0.001, two-way ANOVA test. One representative of 3 experiments is shown.
Fig. 4
Fig. 4. I.v. MOG-induced tolerogenic phenotype of CD11c+CD11b+ DCs is abrogated by injection of clodronate-loaded liposomes
Mice immunized for EAE induction were i.v. injected with MOG or PBS, while PBS-loaded or clodronate-loaded liposomes were i.p. injected. Splenocytes and CNS infiltrating cells were isolated 21 days p.i. and analyzed by flow cytometry. (A) Expression of CD40, CD80, CD86 and IA in gated CD11c+CD11b+ DCs in splenocytes (left) and CNS infiltrating cells (right). (B) Results in (A) were analyzed and shown as mean ± SEM (n=3 each group). *p<0.05, ***p<0.001, two-way ANOVA test. One representative of 3 experiments is shown.
Fig. 5
Fig. 5. iDC-depletion inhibits i.v. MOG-induced IL-27 and TGF-β production
Mice immunized for EAE induction were i.v. injected with MOG or PBS, while PBS-loaded or clodronate-loaded liposomes were i.p. injected. Splenocytes were isolated 21 days p.i. and cultured in triplicate in 24-well plates with MOG35–55 for 24, 48 or 72 hours. (A) Supernatants were collected and analyzed for IL-27and TGF-β production by ELISA and shown as mean ± SEM (n=4 each group). (B) Intracellular secretion of TGF-β and IL-27 in gated CD11c+CD11b+ DCs was determined by flow cytometry. *p<0.05, ***p<0.001, two-way ANOVA test. One representative of 3 experiments is shown.
Fig. 6
Fig. 6. I.v. MOG-induced suppression of Th1/Th17 cells and increase of Treg cells and Tr1 cells is blocked by CD11c+CD11b+ DC depletion
Mice immunized for EAE induction were i.v. injected with MOG or PBS, while PBS-loaded or clodronate-loaded liposomes were i.p. injected. Splenocytes and CNS infiltrating cells were isolated 21 days p.i. (A) Splenocytes triplicate cultured in 24-well plates restimulated with MOG35–55 for 24, 48 or 72 hours and supernatants were collected and analyzed for IL-2, IL-17, and IFN-γ production by ELISA and shown as mean ± SEM (n=4 each group). (B) Intracellular secretion of IL-2, IFN-γ and IL-17 in gated CD4+ T cells in splenocytes (top) and CNS infiltrating cells (bottom) were determined by flow cytometry. (C) Treg cells and Tr1 cells in splenocytes were determinate as CD25+ Foxp3+ and IL-10+ Foxp3− cells, respectively, in gated CD4+ T cells (left). Results were statistically analyzed and data shown as mean ± SEM (n=3 each group) (right). *p<0.05, ***p<0.001,***p<0.001, two-way ANOVA test. One representative of 3 experiments is shown.
Fig. 7
Fig. 7. Effect of iDC depletion on MOG-reactive T cells in co-culture
CD4+ T cells from naïve spleens of MOG TCR transgenic mice (2D2) were co-cultured with DCs from spleen of EAE mice treated with MOG-i.v.+PBS-i.p. (MOG-i.v.+PBS-i.p. DCs & 2D2 CD4) or MOG-i.v.+clodronate-i.p. (MOG-i.v.+clodronate-i.p. DCs & 2D2 CD4) in the presence of MOG35–55 for 3 days. (A) CD11c+ cells were gated and the intracellular secretion of IL-27 and IL-10 in CD11c+CD11b+ cells were analyzed (up) and expressed as mean ± SEM (n=3 each group; bottom). (B) CD4+ cells were gated and their intracellular IFN-γ, IL-17, Foxp3 and IL-10 were analyzed with flow cytometry (top) and data are shown as mean ± SEM (n=3 each group; bottom). (C) Culture supernatants were harvested and production of IL-27, IL-10, IL-2, IFN-γ, IL-17 was analyzed with ELISA. Data are expressed as mean ± SEM (n=4 each group). (D) MOG-induced T-cell proliferative responses were measured by 3H-methylthymidine incorporation and the values of cpm (counts per minute) are shown as mean ±SEM (n=3 each group). *p<0.05, unpaired Student’s t test. One representative of 3 experiments is shown.
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References

    1. Grigoriadis N, van Pesch V. A basic overview of multiple sclerosis immunopathology. Eur J Neurol. 2015;22(Suppl 2):3–13. - PubMed
    1. Hilliard BA, Kamoun M, Ventura E, Rostami A. Mechanisms of suppression of experimental autoimmune encephalomyelitis by intravenous administration of myelin basic protein: role of regulatory spleen cells. Exp Mol Pathol. 2000;68:29–37. - PubMed
    1. t Hart BA, van Kooyk Y, Geurts JJ, Gran B. The primate autoimmune encephalomyelitis model; a bridge between mouse and man. Ann Clin Transl Neurol. 2015;2:581–593. - PMC - PubMed
    1. Rangachari M, Kuchroo VK. Using EAE to better understand principles of immune function and autoimmune pathology. J Autoimmun. 2013;45:31–39. - PMC - PubMed
    1. Luchtman DW, Ellwardt E, Larochelle C, Zipp F. IL-17 and related cytokines involved in the pathology and immunotherapy of multiple sclerosis: Current and future developments. Cytokine Growth Factor Rev. 2014;25:403–413. - PubMed

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