Signaling via the RIP2 adaptor protein in central nervous system-infiltrating dendritic cells promotes inflammation and autoimmunity

Abstract

Peripheral peptidolgycan (PGN) is present within antigen-presenting cells in the central nervous system (CNS) of multiple sclerosis (MS) patients, possibly playing a role in neuroinflammation. Accordingly, PGN is linked with disease progression in the animal model of MS, experimental autoimmune encephalomyelitis (EAE), but the role of specific PGN-sensing proteins is unknown. Here we report that the progression of EAE was dependent on the intracellular PGN sensors NOD1 and NOD2 and their common downstream adaptor molecule, receptor interacting protein 2 (RIP2; also known as RIPK2 and RICK). We found that RIP2, but not toll-like receptor 2 (TLR2), played a critical role in the activation of CNS-infiltrating dendritic cells. Our results suggest that PGN in the CNS is involved in the pathogenesis of EAE through the activation of infiltrating dendritic cells via NOD1-, NOD2-, and RIP2-mediated pathways.

Figure 1

NOD1-, NOD2-, RIP2- and TLR2-deficient mice are resistant to EAE progression. (a-d) Clinical scores of wild-type (WT) mice (n = 13–20) and (a) Nod1^−/− (n = 8), (b) Nod2^−/− (n = 15), (c) Ripk2^−/− (n = 21), (d) Tlr2^−/− (n = 16) mice were determined daily after immunization with MOG (35–55) peptide in CFA. Data are representative of two independent experiments. (e) Clinical scores of WT mice (n = 8) and Ripk2^−/− (n = 9) mice were determined daily after immunization with MOG (35–55) peptide in IFA and soluble peptidoglycan. Data were analyzed using Two-Way ANOVAs. (mean and s.e.m.).

Figure 2

Regulation of EAE development by NOD1, NOD2, RIP2 and TLR2. Histology of paraffin sections of spinal cords isolated from WT, Nod1^−/−, Nod2^−/−, Ripk2^−/− and Tlr2^−/− mice (n = 4 per group) on day 17 after immunization. Arrows indicate axon demyelination. Images are representative of the group. H&E, hematoxylin and eosin; LFB, Luxol fast blue.

Figure 3

NOD1, NOD2, RIP2 and TLR2 are not required for MOG-specific T cell development and proliferation. (a) Quantification of the proliferation of MOG-specific T cells isolated from the spleens of mice 10 days after immunization (n = 7–8). (b) Absolute number of MOG-specific CD4⁺IFNγ⁺ (Th1), CD4⁺TNFα⁺ (Th1) and CD4⁺IL-17⁺ (Th17) T cells in the draining lymph node 10 days after immunization (n = 8). * P < 0.05 and ** P < 0.005, versus WT (Student’s t-test). Data are representative of two independent experiments. (mean and s.e.m.).

Figure 4

CNS accumulation of T cells during EAE is dependent on RIP2. (a) Quantification of CD4⁺ T cells in mononuclear cell infiltrates isolated from spinal cords of mice 17 days after immunization (n = 8). (b) CD3 T cell histology staining of paraffin sections of spinal cords isolated from untreated (UT) mice and mice 17 days after immunization (n = 4). Images are representative of the group. (c) Absolute numbers of MOG-specific CD4⁺IFNγ⁺ (Th1), CD4⁺TNFα⁺ (Th1) and CD4⁺IL-17⁺ (Th17) T cells in the spinal cord 17 days after immunization (n = 8). * P < 0.05, ** P < 0.005 and *** P < 0.001, versus WT (Student’s t-test). Data are representative of two independent experiments. (mean and s.e.m.). See also Figure S1.

Figure 5

Accumulation and activation of CD11c⁺ cells within the CNS during EAE is dependent on RIP2. (a,b) Quantification of (a) CD11c⁺ cells and (b) APC subsets in mononuclear cell infiltrates isolated from spinal cords of mice 17 days after immunization (n = 5–8). (c) Percentage activated of the CD11c⁺ cell population measured by high MHC II surface expression (n = 8). (d) Absolute number of activated CD11c⁺ cells in mononuclear cell infiltrates isolated from spinal cords of mice 17 days after immunization (n = 8). (e) CD11c⁺ cells were isolated from the spinal cords of WT and Ripk2^−/− mice on day 17 of EAE disease progression. Isolated APCs were pooled and cultured with naïve 2D2 TCR T cells (10:1 T cell:APC ratio) for 72 h. Supernatants were collected and IFNγ and IL-17 was measured. (n = 11–14). * P < 0.05, ** P < 0.005 and *** P < 0.001, versus WT (Student’s t-test). Data are representative of two independent experiments. (mean and s.e.m.). See also Fig. S2.

Figure 6

RIP2 signaling is critical for EAE progression via CNS-infiltrating dendritic activation (a) Clinical scores of EAE progression in WT/RIP2 bone marrow chimeric mice (n = 13). Data are representative of two independent experiments. (b) Absolute numbers of CD4⁺ T cells and CD11c⁺ cells in monocuclear cell infiltrates isolated from spinal cords of mice 15 days after immunization (n = 5). (c) Clinical scores of EAE progression following the adoptive transfer of MOG-specific Ripk2^−/− or WT T cells into naïve WT mice (n = 10). (d) Quantification of peripherally-derived dendritic cell and microglia populations, distinguished using congenic surface markers (n = 5). Data were analyzed using a Two-Way ANOVA (a,c) or Student’s t-test (b,d). * P < 0.05, ** P < 0.005 and *** P < 0.001, versus WT (Student’s t-test; b,d). (mean and s.e.m.).