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. 2015 Jan 15;194(2):531-41.
doi: 10.4049/jimmunol.1401320. Epub 2014 Dec 10.

CCR2-dependent dendritic cell accumulation in the central nervous system during early effector experimental autoimmune encephalomyelitis is essential for effector T cell restimulation in situ and disease progression

Benjamin D Clarkson  1 , Alec Walker  2 , Melissa G Harris  2 , Aditya Rayasam  3 , Matyas Sandor  2 , Zsuzsanna Fabry  4
Affiliations

CCR2-dependent dendritic cell accumulation in the central nervous system during early effector experimental autoimmune encephalomyelitis is essential for effector T cell restimulation in situ and disease progression

Benjamin D Clarkson et al. J Immunol. .

Abstract

Dendritic cells (DCs)--although absent from the healthy CNS parenchyma--rapidly accumulate within brain and spinal cord tissue during neuroinflammation associated with experimental autoimmune encephalomyelitis (EAE; a mouse model of multiple sclerosis). Yet, although DCs have been appreciated for their role in initiating adaptive immune responses in peripheral lymphoid organ tissues, how DCs infiltrate the CNS and contribute to ongoing neuroinflammation in situ is poorly understood. In this study, we report the following: 1) CD11c(+) bone marrow-derived DCs and CNS-infiltrating DCs express chemokine receptor CCR2; 2) compared with CCR2(+/+) cells, adoptively transferred CCR2(-/-) bone marrow-derived DCs or DC precursors do not accumulate in the CNS during EAE, despite abundance in blood; 3) CCR2(-/-) DCs show less accumulation in the inflamed CNS in mixed bone marrow chimeras, when compared with CCR2(+/+) DCs; and 4) ablation of CCR2(+/+) DCs during EAE clinical onset delays progression and attenuates cytokine production by infiltrating T cells. Whereas the role of CCR2 in monocyte migration into the CNS has been implicated previously, the role of CCR2 in DC infiltration into the CNS has never been directly addressed. Our data suggest that CCR2-dependent DC recruitment to the CNS during ongoing neuroinflammation plays a crucial role in effector T cell cytokine production and disease progression, and signify that CNS-DCs and circulating DC precursors might be key therapeutic targets for suppressing ongoing neuroinflammation in CNS autoimmune diseases.

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Figures

Figure 1
Figure 1. Ablation of CD11c+ DCs during pre-clinical EAE clinical delays disease progression
A) CD11c-DTR mice were injected with diphtheria toxin (DT) (625ng / kg, i.p.) or PBS control and 24–48 hours later immune cells were isolated from CNS tissues, spleen, and pooled lymph nodes (axial, mesenteric, cervical). CNS tissues were harvested from mice with EAE on day 13 post immunization (receiving i.p. injections of DT on day 7, 9, and 11). Representative dot plots show CD11b and CD11c expression on CD45-high gated leukocytes (above). Numbers indicate percentage of cells in boxed gate. Data representative of 2 independent experiments with n = 3 mice per group. B) Mean clinical scores in CD11c-DTR mice following EAE induction by MOG35-55-immunization. CD11c+ cells were ablated on days 7, 9 and 11 (green lines) or day 14, 16, 18, and 20 (blue lines) by administration of diphtheria toxin (625ng / kg, i.p.). Data representative of 2 independent experiments with n = 3–5 mice per group. *p < 0.05 Student’s t test. Error bars indicate s.e.m.
Figure 2
Figure 2. CD11c+ bone marrow-derived and CNS-infiltrating DCs express CCR2 and CCR2 contributes to BMDC infiltration into the inflamed CNS
A) Migration of GFP+ BMDCs across mouse brain endothelial cell monolayers into lower compartments of single-layer transwell artificial blood brain barrier models in response to dose curve of CCL2. Measurements were made by flow cytometry and quantified relative to number of non-fluorescent loading-control cells (added directly to lower chamber). B) Column graphs show absolute number of CD45-low CD11b+ microglia, infiltrating myeloid cells (CD45-high CD11b+), and dendritic cells (CD45-high CD11b+ CD11c+) in the ipsilateral hrmisphere of mice i.c. injected as indicated. C) CCR2 expression on indicated cell populations isolated from CNS tissues of C57BL6 mice with MOG-induced EAE (DPI 12). CD11c+ cells were gated from CD11b+ population. D) BMDCs generated from CCR2−/−CD11c-eYFP or CCR2+/+CD11c-eYFP mice were transferred (25 ×106, i.p.) into WT C57BL/6 mice with ongoing MOG-induced EAE (DPI 12–13). Histograms show CCR2 expression on BMDCs before transfer. Dot plots show CD11c-eYFP+ cells among BMDCs before transfer and among CD45+ immune cells isolated from spleen and CNS tissue 4 days later (quantified below). Data representative of 2 independent experiments with n = 3–6 mice per group. E) Clinical scores in individual BMDC recipient mice from (C). Frequency of CD11c-eYFP+ cells in CNS of CCR2+/+ BMDC and CCR2−/− BMDC recipients with similar clinical scores. *p < 0.05 Student’s t test. Error bars indicate s.e.m.
Figure 3
Figure 3. CCR2−/− DCs do not accumulate in inflamed CNS despite abundance in blood
CD45.2+ bone marrow cells were adoptively transferred into CD45.1+ hosts at day 12 or day 16 of EAE and mice were euthanized for tissue collection 4 days later at day 16 or day 20, respectively. A) Flow plots showing spleen cells from CD45.1 mice adoptively transferred with either PBS or equal mixtures of bone marrow (BM) from CD45.2+/+ CCR2+/+ DsRed+ and CD45.2+/+ CCR2−/− DsRed+ mice. B) Frequency of CCR2+/+ (Dsred+) and CCR2−/− (Dsred−) among donor (CD45.2+) cells in blood and CNS. Lines indicate difference in frequency in blood and CNS from individual mice. C) Frequency of CCR2+/+ (Dsred+) and CCR2−/− (Dsred−) among donor (CD45.2+) cells in bone marrow, spleen, blood, and CNS at day 16 and 20 of EAE. Data are representative of 3 independent experiments with n = 3 mice per group. Error bars indicate s.e.m. D) Representative flow plots showing percentage of cells CCR2+/+ (Dsred+) and CCR2−/− (Dsred−) among donor (CD45.2+CD45.1−) CD11b+ and CD11c+ cells in blood, spleen, and CNS of mice from (A) Data are representative of 3 independent experiments with n = 3 mice per group.
Figure 4
Figure 4. Deficient CNS accumulation of CCR2−/− CD11c-eYFP+ DCs in mixed bone marrow chimeras
A) Flow plots showing frequency of CD45highCD11c-eYFP+ in cerebellum, spinal cord, and spleen of at day 12 of EAE in mixed BM chimera mice receiving CCR2+/+CD11c-eYFP or CCR2−/−CD11c-eYFP BM cells. B) Total number of CD45-high CD11c-eYFP+ cells and number of microglia (CD11b+CD45-low cells) per gram tissue in the indicated CNS regions at day 12 and day 16 EAE in CCR2+/+CD11c-eYFP and CCR2−/−CD11c-eYFP BM recipients. C) Frequency of CD11c+ cells among CD45+, CD45+CD11b+ and CD45+CD11b− cell populations from spleen and cervical lymph node of CCR2+/+CD11c-eYFP and CCR2−/−CD11c-eYFP BM recipients at day 12 of EAE. D) Fluorescent micrographs from CCR2+/+CD11c-eYFP and CCR2−/−CD11c-eYFP BM recipient chimeric mice depicting CD11c-eYFP+ cell accumulation in cerebellum, olfactory bulb and tissue surrounding the lateral ventricle. Boxes indicate region magnified below. Scale bars 50 microns. *p < 0.05 Student’s t test. Error bars indicate s.e.m.
Figure 5
Figure 5. Ablation of CCR2+/+ DCs during EAE clinical onset delays disease progression
A) Mean clinical scores following MOG35-55-immunization induced EAE shown for groups of WT mice (n = 5) reconstituted with mixtures of BM cells from the indicated donors. Mice were treated with diphtheria toxin (625ng / kg, i.p.) or PBS on DPI 8, 10, and 12. B) Total number (per gram tissue) and frequency of CNS-infiltrating CD4+ and CD8+ T cells expressing the indicated cytokines after 5 hour ex vivo restimulation with PMA / ionomycin in DT treated mice. C) Spinal cord demyelination expressed as percent of total cross-sectional area still myelinated at day 16 EAE (n =5 mice per group). D) Luxol fast blue (LFB) and hematoxylin and eosin (H&E) stained micrographs of spinal cord tissue from DT-treated CCR2+/+:CD11c-DTR and CCR2−/−: CD11c-DTR recipient mixed BM chimeric mice with EAE (DPI 16). Arrows indicate inflammatory foci (outlined with dashed lines, bottom) and demyelinated lesions (outlined with dashed lines, top) *p < 0.05 Student’s t test. Error bars indicate s.e.m.
Figure 6
Figure 6. MHC class II expression on CNS-infiltrating DCs contributes to proliferation, recruitment, and cytokine production by co-infiltrating, myelin specific effector T cells
A) Fluorescent micrographs of DAPI-stained sagittal brain sections showing developing peri-ventricular inflammatory lesions in CD11c-eYFP mice after adoptive transfer of purified CD4+ MOG-specific 2D2.Dsred T cells and EAE induction (DPI 12). CD11c-eYFP+ cells shown in green. 2D2.Dsred T cells shown in red. B) Frequency of MHC class II-expressing cells among various CD11c+ and CD11c− cell subsets isolated from CNS of CD11c-eYFP mice with EAE (DPI 12). C) Mean EAE clinical score is shown for groups (n =3) of WT mice reconstituted with mixtures of BM cells from the indicated donors. Mice were treated with diphtheria toxin (625ng/kg, i.p.) or PBS on DPI 8, 10, 12, and 14. D) Total number (per gram tissue) of spinal cord-infiltrating CD4+ T cells that produced IFNγ or IL-17 following 5 hour ex vivo restimulation with MOG35-55 peptide (20μg/mL). E) Pure FACS-sorted populations of MOG-specific naïve 2D2.Thy1.1 (CD4+ Vβ11+ CD62L+ CD44− Thy1.1+ Dsred−) or antigen-experienced 2D2.Dsred (CD4+ Vβ11+ CD62L− CD44+ Thy1.1− Dsred+) T cells were mixed together, labeled with violet cell trace, and adoptively transferred into congenic MHC II−/−:CD11c-DTR mice with EAE (DPI 9). Recipient mice were treated with either diphtheria toxin (625ng / kg, i.p.) or PBS on DPI 8, 10, 12, and 14. Bar graphs show frequency of proliferated (LFA1-high violet cell trace-low) and unproliferated (LFA1-low violet cell trace-high) transferred 2D2 T cells in cervical lymph node, spleen, and CNS tissues (expressed per gram tissue). Gating of transferred cells is shown in (F). *p < 0.05 Student’s t test. Error bars indicate s.e.m.
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