CC chemokine receptor 4 is required for experimental autoimmune encephalomyelitis by regulating GM-CSF and IL-23 production in dendritic cells

Abstract

Dendritic cells (DCs) are pivotal for the development of experimental autoimmune encephalomyelitis (EAE). However, the mechanisms by which they control disease remain to be determined. This study demonstrates that expression of CC chemokine receptor 4 (CCR4) by DCs is required for EAE induction. CCR4(-/-) mice presented enhanced resistance to EAE associated with a reduction in IL-23 and GM-CSF expression in the CNS. Restoring CCR4 on myeloid cells in bone marrow chimeras or intracerebral microinjection of CCR4-competent DCs, but not macrophages, restored EAE in CCR4(-/-) mice, indicating that CCR4(+) DCs are cellular mediators of EAE development. Mechanistically, CCR4(-/-) DCs were less efficient in GM-CSF and IL-23 production and also T(H)-17 maintenance. Intraspinal IL-23 reconstitution restored EAE in CCR4(-/-) mice, whereas intracerebral inoculation using IL-23(-/-) DCs or GM-CSF(-/-) DCs failed to induce disease. Thus, CCR4-dependent GM-CSF production in DCs required for IL-23 release in these cells is a major component in the development of EAE. Our study identified a unique role for CCR4 in regulating DC function in EAE, harboring therapeutic potential for the treatment of CNS autoimmunity by targeting CCR4 on this specific cell type.

Fig. 1.

CCR4 deficiency in hematopoietic cells confers EAE resistance. (A) Real-time PCR analysis of CCR4 mRNA expression in the CNS of MOG-immunized C57BL/6 mice (n = 3–5 mice per group). mRNA levels are normalized to GAPDH expression, and results are presented as mean ± SEM. (B) Course of active EAE in WT and CCR4^−/− mice (n = 8–11 mice per group; P < 0.001, days 12–22; P < 0.05, days 24–26). (C) Immunohistochemistry of spinal cord sections from WT and CCR4^−/− mice (n = 8 mice per group). (D) Flow cytometry of CNS-isolated mononuclear cells of WT and CCR4^−/− mice. Representative dot plots show percentages of CD4⁺ T cells (CD45^high pregated) in WT and CCR4^−/− mice (mean ± SE; dot blots P < 0.0001; n = 12–13 mice per group). (E) ELISpot analysis of CNS-isolated IL-17–producing MOG-reactive lymphocytes from MOG-immunized WT and CCR4^−/− mice (**P < 0.01). (F) EAE in BM chimeric mice after MOG immunization. Lethally irradiated WT mice were reconstituted with WT or CCR4^−/− BM cells (n = 8–11 mice per group; P < 0.001, days 14–34). (G) EAE in BM chimeric mice after MOG immunization. Lethally irradiated WT or CCR4^−/− mice were reconstituted with WT BM cells (n = 8–11 mice per group). Data (A–E) are representative of at least two independent experiments.

Fig. 2.

CCR4^−/− mice generate encephalitogenic T cells but are resistant to passive EAE. (A) Course of EAE induced in WT mice after adoptive transfer of MOG-reactive WT or CCR4^−/− T lymphocytes (n = 5 mice per group). (B) Course of EAE in WT and CCR4^−/− mice after adoptive transfer of MOG-reactive WT lymphocytes (n = 7 mice per group; P < 0.01, days 14–29). Data shown are representative for at least two independent experiments.

Fig. 3.

CCR4-expressing DCs are mediators of EAE. (A) Course of EAE in mixed BM chimeric mice. Lethally irradiated WT mice reconstituted with CCR4^−/−; mixed CCR4^−/− and RAG-2^−/−; mixed CCR4^−/− and RAG-2-cγc^−/−; or WT BM cells (n = 12–13 mice per group) were MOG immunized after 7–8 wk. (B) Clinical scores for CCR4^−/− mice injected with CCR4^+/+ MOG DCs or CCR4^+/+ MOG macrophages and CCR4^−/− control mice after MOG immunization (n = 6–8 mice per group). Data shown are representative of at least two independent experiments.

Fig. 4.

Reduced IL-23 production and maintenance of T_H-17 cells in CCR4^−/− DCs. (A) ELISA of IL-23 production in WT or CCR4^−/− BMDCs after stimulation with TLR ligands. Shown are mean ± SEM of cytokine levels in culture supernatants; n = 8 mice per group. (B) Percentages of IL-17–producing MOG-reactive CD4⁺ T cells after in vitro rechallenge with MOG-loaded WT DCs or CCR4^−/− DCs or PMA/ionomycin. Diagram shows mean IL-17–producing cells ± SE; n = 4–5 MOG-immunized mice per group. (C) ELISA of IL-23 in the WT and CCR4^−/− CNS at peak of disease. Mean protein amount per CNS ± SEM; n = 3–4 mice per group. (D) Flow cytometry of IL-23–producing CNS invading DCs of MOG-immunized WT and CCR4^−/− mice at peak disease. Representative histograms show percentages of IL-23–producing DCs (CD45^high CD11c⁺ pregated) in the WT or CCR4^−/− CNS. Mean ± SEM; n = 5–6 mice per group; P < 0.01. (E) Clinical scores for CCR4^−/− mice injected i.c. with IL-23^−/− MOG-loaded DCs, CCR4^+/+ MOG-loaded DCs, or CCR4^−/− MOG-loaded DCs (n = 5–6 mice per group). (F) Clinical scores for CCR4^−/− mice injected i.c. with IL-23 (days 8–9 after MOG immunization) or MOG-immunized WT controls (n = 6–8 mice per group; P < 0.05, days 14; P < 0.01, days 16–24; for i.c. 75 ng IL-23 CCR4^−/− vs. WT mice). Data are representative of at least two independent experiments; **P < 0.01; *P < 0.05.

Fig. 5.

CCR4 ligands induce IL-23 release in CCR4⁺ DCs via a GM-CSF–dependent pathway. (A) GM-CSF release in BMDCs from WT or CCR4^−/− mice after CCL17/CCL22 stimulation (n = 5 mice per group; mean protein amount ± SEM). (B) GM-CSF levels in the CNS of WT and CCR4^−/− mice at peak of disease. Mean protein amount/CNS ± SEM; n = 3–4 mice per group. (C) Representative dot plots of GM-CSF production in CD45^high CNS-invading DCs in WT and CCR4^−/− mice. (D) ELISA of GM-CSF produced by CNS mononuclear cells of WT and CCR4^−/− mice after LPS stimulation. Mean protein amount ± SEM; n = 5–6 mice per group. (E) IL-23 release by WT or CCR4^−/− BMDCs after LPS stimulation without or with GM-CSF. Mean protein amount ± SEM; n = 5 mice per group; ***P < 0,001. (F) IL-23 release by C57BL/6; C57BL/6 treated with anti-CCL17/22; CCR4^−/−; and GM-CSF^−/− DCs with or without LPS. Mean protein amount ± SEM; n = 4–5 mice per group. **P < 0.01, ***P < 0,001 between indicated group compared with C57BL/6 DCs. (G) Clinical scores for CCR4^−/− mice i.c. injected with GM-CSF^−/− DCs or MOG-immunized WT or CCR4^−/− mice (n = 7–8 mice per group; P < 0.05, day 12; P < 0.01, day 14; P < 0.001, days 16–18). Data are representative of at least two independent experiments.