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. Author manuscript; available in PMC: 2012 Jan 28.
Published in final edited form as: Immunity. 2011 Jan 13;34(1):75–84. doi: 10.1016/j.immuni.2010.12.015

RIP2 signaling in Central Nervous System-Infiltrating Dendritic Cells Promotes Cellular Activation, Inflammation and Autoimmune Disease Progression

Patrick J Shaw 1, Maggie J Barr 1, John R Lukens 1, Maureen A McGargill 1, Hongbo Chi 1, Tak W Mak 2, Thirumala-Devi Kanneganti 1
PMCID: PMC3057380  NIHMSID: NIHMS262696  PMID: 21236705

Summary

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.

Keywords: NLR, RIPK2, RIP2, NOD1, NOD2, peptidoglycan, PGN, EAE, multiple sclerosis

Introduction

Multiple sclerosis (MS) affects more than 2.1 million people worldwide, and is the leading cause of neurological disability in young adults (McFarland and Martin, 2007). MS is an inflammatory, demyelinating disease in which autoimmune T cells are directed against antigens that are derived from the central nervous system (CNS) (Sospedra and Martin, 2005). However, the presence of myelin-reactive T cells have been found in healthy individuals suggesting that other factors play an important role in initiation and perpetuation of disease progression (Compston and Coles, 2002). Bacterial and viral infections are potential cofactors implicated in the initiation and persistence of autoimmune diseases (Wucherpfennig, 2001). Accordingly, MS relapses and their severity are frequently associated with antecedent viral and bacterial infections (Buljevac et al., 2002; Buljevac et al., 2003; Sibley et al., 1985).

The mechanisms underlying MS have been widely studied using the animal model experimental autoimmune encephalomyelitis (EAE). EAE is induced in mice by immunization with myelin oligodendrocyte glycoprotein (MOG) peptide in complete Freund’s adjuvant (CFA) containing heat killed M. Tuberculosis (Stromnes and Goverman, 2006a). It is believed that genetically predisposed individuals develop MS following an environmental trigger that activates myelin-specific T cells. The initial activation allows the T cells to cross the blood-brain barrier, but once in the CNS the T cells must be reactivated by antigen-presenting cells (APCs) (Goverman, 2009). The presence of peptidoglycan (PGN)-containing APCs was detected in the brains of multiple sclerosis patients, but not in healthy controls during a postmortem analysis (Schrijver et al., 2001). Additionally, PGN-containing APCs were detected and localized to CNS lesions in both non-human primate (Visser et al., 2006) and mouse (Visser et al., 2005) models of MS, but were not detected in healthy control animals. Furthermore, EAE can be induced in mice when the bacterial cell wall component, PGN, is used as the adjuvant in place of CFA (Visser et al., 2005). Therefore, the presence of PGN in the inflamed brain appears to play a role in the progression of EAE, especially considering the association between bacterial infection and MS (Buljevac et al., 2003). However, the role of PGN-activated signaling pathways in EAE progression has not been explored.

Recognition of PGN by both extracellular and intracellular pattern recognition receptors induces innate immune responses. The transmembrane protein toll-like receptor 2 (TLR2) can be activated by PGN (Dziarski and Gupta, 2005). TLR2 has been reported to play a role in the progression of EAE; however, the mechanism reported was independent of PGN (Farez et al., 2009). Additionally, members of the Nod-like receptor (NLR) family, NOD1 and NOD2, detect PGN fragments in the cytosol (Chamaillard et al., 2003; Girardin et al., 2003). Activation of NOD1 and NOD2 initiates a pro-inflammatory signaling cascade dependent on the recruitment of their common adaptor protein, receptor-interacting protein 2 (RIP2; also known as RIPK2 and RICK) and NF-κB activation (Inohara et al., 2000; Park et al., 2007). Furthermore, RIPK2 was one of a small number of genes, which was upregulated in non T-cell fractions of the peripheral blood of MS patients (Satoh et al., 2005). However, the role of NOD1, NOD2 or RIP2 in EAE disease progression has not been determined

In this study, we demonstrate that TLR2, NOD1, NOD2 and RIP2 play a critical role in the progression of EAE. The development and proliferation of MOG-specific T cells in lymphoid organs was not reduced in any of the gene-targeted mice, but the number of T cells in the CNS was reduced in Nod1−/−, Nod2−/− and RIP2-deficient (Ripk2−/−) mice. Additionally, the number of activated dendritic cells in the CNS following MOG-induced EAE was greatly reduced in Nod1−/−, Nod2−/− and Ripk2−/− mice, but not Tlr2−/− mice. This decrease in CNS-infiltrating APC activation resulted in a functional defect in the ability of APCs to drive the differentiation of naïve MOG 35–55 specific TCR (2D2) T cells into T helper 17 (Th17) effector cells. Furthermore, WT mice reconstituted with Ripk2−/− bone marrow were resistant to EAE. This suggests that RIP2 plays a role in EAE progression, which is intrinsic to T cells or peripheral antigen-presenting cells. However, the adoptive transfer of MOG-specific Ripk2−/− T cells caused disease progression to a similar degree as MOG-specific WT T cells, eliminating the possibility of a T cell-intrinsic role for RIP2 in EAE progression. These findings suggest a mechanistic pathway by which antigen-presenting cells that infiltrate the CNS are activated by intracellular PGN through NOD1 and NOD2, and in turn RIP2, leading to neuroinflammation and the reactivation and expansion of MOG-specific T cells to drive EAE pathogenesis.

Results

Nod1−/−, Nod2−/− and Ripk2−/− mice are highly resistant to EAE

To study the potential role of PGN-sensing pattern recognition receptors in the progression of EAE, we compared the susceptibility and severity of WT, Nod1−/−, Nod2−/−, Ripk2−/− and Tlr2−/− mice to EAE induction. When immunized by subcutaneous injection of a MOG peptide emulsified in complete Freund’s adjuvant and accompanied by pertussis toxin, WT mice developed disease as characterized by increasing paralysis. Notably, Nod1−/−, Nod2−/− and Ripk2−/− mice were protected from the progression of EAE as measured by clinical scores (Fig. 1a–c). Additionally, Tlr2−/− mice were resistant to EAE (Fig 1d), as reported previously (Farez et al., 2009). The incidence of disease following EAE induction was similar between WT and the gene-deficient mice; however the severity of disease progression was dampened in all groups of gene-deficient mice (Table 1). The percent of mice that developed hind-limb paralysis following EAE immunization was reduced in all gene-deficient mice (Table 1). However, protection from disease progression was more consistent in Ripk2−/− mice when compared to Nod1−/− or Nod2−/− mice. Additionally, the day of onset was slightly delayed in Nod1−/−, Nod2−/− and Ripk2−/− mice. Interestingly, the Tlr2−/− mice actually had a quicker onset of disease despite being protected from disease later in the timecourse. This finding is similar to previously reported EAE clinical scores in Tlr2−/− mice (Farez et al., 2009; Prinz et al., 2006). Histological analysis of spinal cord sections also showed that WT mice developed prominent inflammatory infiltration and axon demyelination (Fig. 2). In contrast, Nod1−/−, Nod2−/− and Ripk2−/− mice had decreased inflammatory infiltrate and decreased axon demyelination. Tlr2−/− mice exhibited similar inflammatory infiltration compared to WT mice, but had decreased axon demyelination.

Figure 1.

Figure 1

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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.).

Table 1.

EAE progression in wild-type, Nod1−/−, Nod2−/−, Ripk2−/− and Tlr2−/− mice

Mouse genotype Incidence Day of onset (mean ± s.e.m.) Developed complete hind-limb paralysis (%) Maximum clinical score (mean ± s.e.m.)
Wild-type 50/51 (98.03%) 14.10 ± 0.31 95 2.83 ± 0.24
Nod1−/− 8/8 (100%) 16.75 ± 0.86 50 1.31 ± 0.25
Nod2−/− 15/15 (100%) 16.20 ± 0.69 40 1.57 ± 0.19
Ripk2−/− 15/16 (93.75%) 17.67 ± 1.19 19 1.41 ± 0.23
Tlr2−/− 22/22 (100%) 11.68 ± 0.36 55 2.23 ± 0.17
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Results are cumulative data from six different experiments.

Figure 2.

Figure 2

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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.

To determine if the protection from EAE observed in Ripk2−/− mice was PGN-dependent, mice were immunized with MOG peptide and incomplete Freund’s adjuvant containing soluble PGN. Ripk2−/− mice were protected from disease progression following PGN EAE immunization when compared to WT mice (Fig. 1e). These results indicate a critical, PGN-dependent role for NOD1, NOD2 and RIP2 in EAE pathogenesis. Therefore, we further characterized disease progression to identify a potential mechanism by which NOD1, NOD2 and RIP2 contribute to autoimmunity.

NOD1, NOD2 and RIP2 are not required for MOG-specific T cell development and proliferation

To investigate whether NOD1, NOD2, RIP2 and TLR2 contribute to the priming, differentiation and proliferation of MOG-specific T cells, we assessed the number and proliferative capacity of MOG-specific T cells in the peripheral lymphoid organs 10 days after immunization. Splenocytes were incubated with various concentrations of MOG peptide for 48 h, with [H3] Thymidine added for the final 8 h and its incorporation measured. At all concentrations of MOG peptide, the proliferation of MOG-specific T cells was similar between WT and gene-deficient mice (Fig 3a). In addition, we quantified the number of MOG-specific T cells in the draining lymph nodes by stimulating T cells with MOG peptide for 5 h in the presence of monensin and staining for intracellular cytokines. There was not a difference in the number of Th1 or Th17 MOG-specific T cells in Nod1−/−, Nod2−/− or Ripk2−/− mice (Fig. 3b). In contrast, Tlr2−/− mice had substantially more Th1 MOG-specific T cells, which suggests a distinct role for TLR2 in Th1 and Th17 cells, as recently reported (Reynolds et al., 2010), and might explain the earlier disease onset. Nevertheless, our results demonstrate that the resistance to EAE in Nod1−/−, Nod2−/− and Ripk2−/− mice is not due to a defect in MOG-specific T cell development and expansion.

Figure 3.

Figure 3

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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.).

Reduced T cell accumulation in the CNS of Ripk2−/− mice

Lymphocytes were isolated from the CNS of mice and the total number of T cells and MOG-specific T cells were quantified. The number of CNS-infiltrating CD4+ T cells in untreated WT and Ripk2−/− mice is equivalent (Fig. S1a). However, 17 days after EAE immunization, there was a trend towards a reduction in total CD4+ T cells in Nod1−/− and Nod2−/− mice, and Ripk2−/− mice had a significant (p = 0.025) reduction in the total number of CD4+ T cells in the CNS (Fig. 4a). This finding was confirmed with CD3+ T cell immunohistochemistry of spinal cords (Fig. 4b). Nod1−/−, Nod2−/− and Ripk2−/− mice had a drastic reduction in positively stained T cells present in the spinal cord compared to WT mice. To determine if this finding was also reflected in disease-specific T cells, lymphocytes were cultured with MOG peptide for 5 h in the presence of monensin and stained for intracellular cytokines. Ripk2−/− mice had significantly less Th1 (p = 0.031) and Th17 (p = 0.048) MOG-specific CD4+ T cells in the CNS (Fig. 4c). Tlr2−/− mice had similar numbers of MOG-specific T cells as WT mice. The results collectively indicate that RIP2 is involved in a mechanism that affects the CNS accumulation of all T cells, and is not specific to Th1 or Th17 T cells. However, at an early time point (5 days after EAE immunization), WT and Ripk2−/− mice had equivalent numbers of CNS-infiltrating CD4+ T cells (Fig. S1b). Therefore, we conclude that the decrease in CD4+ T cells in the CNS of Ripk2−/− mice at a later time point during EAE is not due to a defect in the ability of lymphocytes to migrate into the CNS.

Figure 4.

Figure 4

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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.

The number of activated of APCs in the CNS is reduced in Nod1−/−, Nod2−/− and Ripk2−/− mice

The progression of EAE is dependent on the reactivation of T cells within the CNS by dendritic cells. In untreated mice, the number of CD11c+ cells is equivalent in WT and Ripk2−/− mice (Fig. S1a). Following EAE induction, cells isolated from the CNS and were analyzed for the number and activation status of dendritic cells. The total number of CD11c+ cells in the CNS 17 days after EAE immunization was reduced in Nod1−/− and Nod2−/− mice, and to an even greater degree in Ripk2−/− mice (Fig. 5a). Of the CNS-infiltrating APCs during EAE, myeloid dendritic cells (mDCs) are the most abundant and critically important to disease progression among the various APC subsets (Bailey et al., 2007; Miller et al., 2007). The CNS-infiltration of mDCs was drastically reduced in Ripk2−/− mice (Fig. 5b). Additionally, the total number of CD8+ DCs and macrophages in the CNS was reduced in Ripk2−/− mice 17 days after EAE induction.

Figure 5.

Figure 5

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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.

To test if the decrease in CD11c+ cells present in the CNS of Ripk2−/− mice is an intrinsic defect in dendritic cell migration, we tested the migratory ability of bone-marrow derived dendritic cells (BMDCs) to chemokine in vitro. Ripk2−/− BMDCs were able to migrate across a transwell to the chemokine gradient to the same degree as WT BMDCs (Fig. S2a). Additionally, we analyzed the infiltration of dendritic cells, macrophages and granulocytes into the CNS 5 days after immunization. At this early timepoint, WT and Ripk2−/− mice have equivalent numbers of CNS-infiltrating dendritic cells, macrophages and granulocytes (Fig. S1b). Therefore, we conclude that the decrease in CD11c+ cells present in the CNS of Ripk2−/− mice at day 17 during EAE is not due to an intrinsic defect in dendritic cell migration.

We next determined if the activation state and functionality of CNS-infiltrating DCs is affected by NOD1-, NOD2- or RIP2-deficiency. CNS-infiltrating DCs express Nod1, Nod2 and Ripk2, with the expression of Nod2 being slightly higher than Nod1 (Fig. S2b). However, we only found that the percent of activated CD11c+ cells, as determined by high MHC II surface expression, was decreased in Ripk2−/− mice (Fig. 5c). The percent of activated CD11c+ cells was slightly decreased in mice deficient in NOD1 or NOD2 alone, but not to a significant degree (p = 0.18, 0.13; respectively). Only in Ripk2−/− mice, equivalent to the removal of both NOD1 and NOD2, is there a significant decrease in CD11c+ activation (p = 0.017). However, the total number of activated CD11c+ cells in the CNS was reduced in Nod1−/− and Nod2−/− mice, and to an even greater degree in Ripk2−/− mice (Fig. 5d). In contrast, Tlr2−/− mice had similar CD11c+ cell numbers and activation levels as WT mice.

The functional ability of APCs isolated from WT and Ripk2−/− spinal cords during EAE progression was examined by culturing the isolated APCs with naïve 2D2 TCR T cells as described previously (Bailey et al., 2007). CNS-infiltrating APCs from WT and Ripk2−/− mice induced the proliferation of naïve 2D2 T cells to a similar degree (data not shown). However, CNS-infiltrating APCs from Ripk2−/− mice were impaired in their ability to drive the differentiation of naïve 2D2 T cells into Th17 effector cells, as measured by cytokine release (Fig. 5e). Overall, these results suggest that NOD1 and NOD2 have redundant roles in CNS-infiltrating dendritic cell activation during EAE. Additionally, RIP2 signaling is critical to CNS-infiltrating dendritic cell activation and, in turn, the ability of these APCs to drive the differentiation of CNS-infiltrating T cells into functional Th17 effector cells, which are required for the progression of EAE and MS.

Resistance to EAE in Ripk2−/− mice is intrinsic to CNS-infiltrating dendritic cell activation

To further examine the particular cell type that requires RIP2 activity for EAE progression, we created Ripk2−/− and WT bone marrow chimeras. As a positive control, Ripk2−/− host mice transplanted with Ripk2−/− bone marrow (Ripk2−/− ≫ Ripk2−/−) were resistant to MOG-induced EAE (Fig. 6a). Irradiated WT mice transplanted with Ripk2−/− bone marrow (Ripk2−/− ≫ WT) were also resistant to EAE, whereas host Ripk2−/− mice transplanted with WT bone marrow developed EAE progression similar to WT mice. This result indicates that RIP2 in the hematopoietic cell population plays a critical role in the development of EAE. Cell populations within the CNS were analyzed 15 days after immunization. Both Ripk2−/− ≫ WT and Ripk2−/− ≫ Ripk2−/− mice had less CD4+ T cells and a trend towards less CD11c+ cells compared to WT ≫ WT mice (Fig. 6b).

Figure 6.

Figure 6

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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.).

Ripk2−/− ≫ WT mice, resistant to EAE progression, have Ripk2−/− peripheral dendritic cells and Ripk2−/− T cells. Therefore, to determine which cell population requires RIP2 signaling for EAE progression we passively induced EAE via an adoptive transfer of MOG-specific T cells into naïve WT mice (Stromnes and Goverman, 2006b). The adoptive transfer of MOG-specific Ripk2−/− T cells induced disease progression to the same degree as WT T cells (Fig 6c). This suggests that EAE progression is not dependent on RIP2 activation in T cells. Collectively with the bone marrow chimera results, these results suggest that EAE progression is dependent on RIP2 activation in peripheral antigen-presenting cells.

Using congenically marked mice for bone marrow chimeras allowed for us to distinguish infiltrating peripheral dendritic cells from CNS-resident microglia. Microglia are irradiation-resistant and only a small fraction are replaced by injected bone marrow in irradiated mice (Flugel et al., 2001; Priller et al., 2001). The activation of infiltrating dendritic cells is dependent on RIP2 (Fig. 6d, top panel). Additionally, microglia activation is dependent on RIP2 (Fig. 6d, bottom panel). Both microglia and infiltrating dendritic cells are activated through a RIP2-mediated pathway, but the majority of CD11c+ cells in the CNS during EAE are peripheral dendritic cells, explaining why only Ripk2−/− ≫ WT mice are resistant to EAE. Collectively, these results indicate that the activation of CNS-infiltrating peripheral dendritic cells is dependent on RIP2, a mechanism likely responsible for the resistance to EAE of Ripk2−/− mice.

Discussion

Antigen-presenting cells (APCs) within the CNS of patients with multiple sclerosis contain PGN, which could potentially stimulate innate immune responses and contribute to CNS autoimmune disease progression in the absence of bacterial replication in the brain (Schrijver et al., 2001). In this study we have investigated the function of cytosolic PGN sensors NOD1 and NOD2, in addition to their common downstream adaptor protein, RIP2, in the progression of EAE.

We have shown here that Nod1−/−, Nod2−/− and Ripk2−/− mice were highly resistant to EAE in a PGN-dependent manner, with Ripk2−/− mice showing the greatest degree of protection. This protection occurs despite normal development of MOG-specific Th1 and Th17 responses in the periphery. Recent studies identified a T cell-intrinsic role for NOD2 in Th1 differentiation during Toxoplasma gondii infection (Shaw et al., 2009). However, NOD1, NOD2 and RIP2 do not play a role in Th1 or Th17 differentiation during EAE, as the development of MOG-specific IFNγ+, TNFα+ and IL-17+CD4+ T cells in the lymph node was similar to WT mice. T cell responses have been shown by several reports to be normal in Ripk2−/− mice (Fairhead et al., 2008; Hall et al., 2008; Nembrini et al., 2008), but it has been suggested that RIP2 is involved in the CD40 signaling pathway, which is critical to EAE development (Du et al., 2009). However, Ripk2−/− mice did show signs of EAE pathogenesis, although to a lesser degree than WT, and had normal MOG-specific T cell development; both of which are in contrast to Cd40−/− mice (Becher et al., 2001).

In contrast to the periphery, there was a decrease in the total number of MOG-specific Th1 and Th17 cells in Ripk2−/− mice in the CNS at day 17 during EAE. However, the accumulation of T cells in the CNS during EAE in Ripk2−/− mice was normal at an early timepoint (day 5). Furthermore, the adoptive transfer of MOG-specific Ripk2−/− T cells induced disease progression to a similar degree as WT T cells. This suggests that RIP2 does not play a role in EAE progression intrinsic to T cell function. Additionally, the reduced accumulation of T cells in the CNS at day 17 in Ripk2−/− mice is not intrinsic to T cell migration, but rather the difference in the CNS accumulation of T cells at day 17 is intrinsic to dendritic cells via reduced reactivation and expansion of T cells in the CNS during EAE.

Similar to CD4+ T cell accumulation, in Ripk2−/− mice the total number of CNS-infiltrating dendritic cells was drastically reduced at day 17 during EAE progression, but was not different from WT mice at an early timepoint (day 5). This, in addition to the normal migration of RIP2-deficient BMDCs, suggests that the RIP2 signaling pathway is not involved in the initial migration of CD11c+ cells into the CNS during EAE. Rather, the reactivation of APCs within the CNS could result in decreases in the accumulation of inflammatory cells during EAE via reduced neuroinflammation and lymphocyte reactivation within the CNS. The percent of CD11c+ cells activated within the CNS during EAE was substantially reduced in Ripk2−/− mice. NOD1 and NOD2 likely have some redundancy in their role during EAE progression, as Nod1−/− and Nod2−/− mice were both resistant to EAE progression. However, the protection from EAE in either Nod1−/− or Nod2−/− mice was less consistent than in Ripk2−/− mice. Additionally, NOD1 or NOD2 deficiency resulted in a trend towards reduced dendritic cell activation in the CNS, whereas mice deficient in RIP2, the common downstream adaptor of NOD1 and NOD2, exhibited a greater, significant reduction in dendritic cell activation. However, both Nod1−/− and Nod2−/− mice were resistant to EAE, in contrast to a report in an arthritis model where Nod1−/− and Nod2−/− mice had opposite phenotypes (Joosten et al., 2008). NOD1 and NOD2 have some redundant role in the activation of dendritic cells within the CNS, but both receptors appear to be dependent on RIP2 for this function.

In Nod1−/−, Nod2−/− and Ripk2−/− mice, CD11c+ activation in the periphery was normal, whereas activation was decreased in the CNS. This is likely due to the very different microenvironments in which the CD11c+ cells are present. CD11c+ cells in the periphery are exposed to a plethora of activating ligands, with the most important likely being extracellular TLR ligands, as evidenced by the inability of MyD88−/− mice to develop EAE (Prinz et al., 2006). Therefore, peripheral Ripk2−/− APCs are activated to the same degree as WT mice, as there are several stimuli present which act independently of RIP2 to activate APCs. In the periphery, dendritic cells phagocytose PGN from either the intestinal microbiota or the CFA in the case of EAE induction (Visser et al., 2005). Once the APCs migrate to within the CNS, extracellular stimuli that can activate APCs are likely less abundant due to the blood brain barrier. Therefore, once APCs migrate to the CNS they become critically dependent on “self-activation” by sensing the intracellular PGN that they have carried into the CNS. Activation of CNS-infiltrating APCs in Tlr2−/− mice were not reduced suggesting the mechanisms underlying resistance to EAE in Ripk2−/− and Tlr2−/− mice are different, therefore inhibition of both RIP2 and TLR2 signaling would likely have an additive effect of protection from EAE. However, the recognition of intracellular PGN, and its inflammatory effect, is dependent on the NOD1-NOD2-RIP2 signaling pathway; therefore, CNS-infiltrating APCs deficient in the RIP2 signaling axis are unable to activate and expand.

Peripherally-derived myeloid dendritic cells present myelin antigens to pre-activated myelin-specific T cells and naïve T cells more efficiently than CNS-resident APCs, microglia (Miller et al., 2007). APCs isolated from the CNS of Ripk2−/− mice were functionally defective in driving the differentiation of naïve 2D2 T cells into effector Th17 cells. Therefore, reduced activation of APCs within the CNS resulted in decreased T cell reactivation and ameliorated EAE progression. Nevertheless, to differentiate the role of RIP2 signaling in peripherally-derived dendritic cells and microglia in the CNS during EAE, WT-Ripk2−/− bone marrow chimeras were created. Microglia are irradiation-resistant and only a small fraction of these cells are readily replenished after bone-marrow transplantation (Flugel et al., 2001; Priller et al., 2001). Therefore, microglia and peripherally-derived dendritic cells can be distinguished. The activation of both microglia and peripherally-derived dendritic cells was dependent on RIP2. However, microglia are inefficient in myelin-specific antigen presentation and peripherally-derived dendritic cells make up the majority of CD11c+ cells in the CNS during EAE. Therefore, only chimeric mice with Ripk2−/− infiltrating dendritic cells are resistant to EAE.

In summary, our observations have provided a mechanistic link by which PGN present in the brain of MS patients could contribute to disease progression. Our results suggest that activation of CNS-infiltrating dendritic cells by PGN is dependent on NOD1- and NOD2-mediated RIP2 activation. The RIP2 signaling pathway was not involved in the migration of T cells and dendritic cells into the CNS during EAE, but rather played a critical role in the “self-activation” of CNS-infiltrating dendritic cells. This “self-activation” of APCs is essential for EAE progression through the reactivation and expansion of MOG-specific T cells. Accordingly, CNS-infiltrating CD11c+ cells isolated from Ripk2−/− mice following EAE immunization functionally impaired in their ability to drive Th17 responses compared to their WT counterpart. Furthermore, RIP2 played a role in the progression, not the initiation, of EAE; therefore RIP2 inhibition might prove therapeutically useful for the alleviation of ongoing disease by preventing T cell reactivation in the CNS. Importantly, RIP2 inhibition would target the dendritic cell component of MS progression, whereas most current MS therapies target the T cell component. Therefore, RIP2 inhibition could provide an additive or synergistic therapeutic effect when used in conjunction with current MS treatment strategies. Overall, inhibition of RIP2 activation in CNS-infiltrating dendritic cells represents a unique and valuable therapeutic strategy for multiple sclerosis.

Experimental Procedures

Mice

C57BL/6 mice were purchased from Jackson Laboratory. The generation of Nod1−/−, Nod2−/−, Ripk2−/− and Tlr2−/− mice have been described previously (Chamaillard et al., 2003; Kobayashi et al., 2002; Kobayashi et al., 2005; Wooten et al., 2002) and backcrossed onto a C57BL/6 background for at least ten generations. Mice were housed in a pathogen-free facility and the animal studies were conducted under protocols approved by St. Jude Children’s Research Hospital Committee on Use and Care of Animals.

EAE model

Female mice 7–9 weeks of age were immunized subcutaneously with 100 μg MOG (35–55) peptide (MEVGWYRSPFSRVVHLYRNGK) emulsified in CFA or incomplete Freund’s adjuvant with soluble PGN (25 μg total per mouse) on day 0. Pertussis toxin (200 ng; List Biological Laboratories, Inc.) was injected intraperitoneally on days 0 and 2 (Stromnes and Goverman, 2006a; Visser et al., 2005). EAE was induced by the adoptive transfer of MOG-specific T cells as described previously (Stromnes and Goverman, 2006b). Briefly, splenocytes were collected from mice 10 days after immunization and cultured for 72 h in the presence of MOG peptide. 15 × 106 T cells were injected intravenously to sublethally irradiated (400 rad) naive WT mice. Pertussis toxin was injected intraperitoneally on days 0 and 2. Disease severity was assessed daily by assigning scores on the following scale: 0, no disease; 1, tail paralysis; 2, weakness of hind limbs; 3, paralysis of hind limbs; 4, paralysis of hind limbs and severe hunched posture; 5, moribund or death. CNS lymphocytes were collected as described previously (Ivanov et al., 2007). Briefly, mice were perfused through the left ventricle with ice cold 2 mM EDTA in PBS. The spinal cord was dissected, cut into small pieces and digested in collagenase D (1 mg/mL; Roche Diagnostics) for 45 min. at 37° C. Spinal cord sections were passed through a 70 μm cell strainer, washed once in PBS, placed in a 38% Percoll solution and pelleted for 20 min. at 2000 rpm. Pellets were resuspended in media and used for subsequent studies and analysis.

MOG-specific T cell proliferation

Cells were harvested from the spleen 10 days following immunization. 2 × 105 cells were plated in triplicate in a 96-well plate with various concentrations of MOG peptide and incubated at 37° C for 48 h. [3H]Thymidine was added for the last 8 h of culture, and the amount of incorporated [3H]thymidine was measured.

Flow cytometry and in vitro stimulation

For the analysis of mouse phenotypes, the following monoclonal antibodies were used: CD4 (L3T4), IFNγ (XMG1.2), IL-17A (eBio17B7), MHC II (M5/114.15.2), CD11b (M1/70), PDCA-1 (eBio927), B220 (RA3-6B2), Gr-1 (RB6-8C5) from eBioscience and TCRβ (H57-597), TNFα (MP6-XT22), CD11c (N418), CD45.1 (A20), CD45.2 (104) from Biolegend. For intracellular cytokine staining, cells were stimulated for 4.5 h with 30 μg/mL MOG peptide, with the final 2 h of culture in the presence of monensin (eBioscience). Following surface staining, cells were permeabilized using Permeabilization wash buffer (Biolegend) and stained for intracellular cytokines. Samples were acquired on a FACSCalibur LSR II (BD Biosciences) and analyzed with Flowjo software (TreeStar).

Activation of 2D2 TCR T cells by CNS-infiltrating APCs

Infiltrating cells were isolated from spinal cords of WT and Ripk2−/− mice on day 17 following EAE immunization. Cells were pooled for each group and CD11c+ cells were purified. Naïve (CD4+CD62L+) 2D2 TCR T cells were cultured with the isolated CNS-infiltrating APCs at a 10:1 ratio. Supernatants were removed after 72 h and IFNγ and IL-17 were measured using a bioplex kit (Millipore).

Histopathology

Mice were euthanized 17 days following MOG immunization. Spinal cords were dissected and fixed in formalin. Spinal cord sections were stained with hematoxylin and eosin, luxol fast blue or polyclonal CD3. Images representative of the group are shown.

Generation of bone marrow chimeras

WT and Ripk2−/− mice, lethally irradiated with a split dose of 1200 rads, were injected with 5 × 106 bone marrow cells isolated from WT or Ripk2−/− mice. The mice were rested for 6 weeks to allow for successful engraftment. The bone marrow transplants were validated using congenic markers (CD45.1 or CD45.2).

Statistics

Differences between clinical score data sets were analyzed by Two-Way ANOVA. Differences between all other data sets were analyzed by Student’s t-test.

Supplementary Material

01

Acknowledgments

We thank R. Flavell (Yale University School of Medicine), T. Mak (University of Toronto) and S. Akira (Osaka University) for the generous supply of mutant mice; and D. Perera and G. Johnson for technical assistance. This work was supported by grants from the National Institutes of Health grant number AR056296 and AI088177, the American Lebanese and Syrian Associated Charities to T-D.K.

Footnotes

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