NLRP3 inflammasome induces chemotactic immune cell migration to the CNS in experimental autoimmune encephalomyelitis

Abstract

The NLRP3 inflammasome is a multiprotein complex consisting of three kinds of proteins, NLRP3, ASC, and pro-caspase-1, and plays a role in sensing pathogens and danger signals in the innate immune system. The NLRP3 inflammasome is thought to be involved in the development of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). However, the mechanism by which the NLRP3 inflammasome induces EAE is not clear. In this study, we found that the NLRP3 inflammasome played a critical role in inducing T-helper cell migration into the CNS. To gain migratory ability, CD4⁺ T cells need to be primed by NLRP3 inflammasome-sufficient antigen-presenting cells to up-regulate chemotaxis-related proteins, such as osteopontin, CCR2, and CXCR6. In the presence of the NLRP3 inflammasome, dendritic cells and macrophages also induce chemotactic ability and up-regulate chemotaxis-related proteins, such as α4β1 integrin, CCL7, CCL8, and CXCL16. On the other hand, reduced Th17 cell population size in immunized Nlrp3^−/− and Asc^−/− mice is not a determinative factor for their resistance to EAE. As currently applied in clinical interventions of MS, targeting immune cell migration molecules may be an effective approach in treating MS accompanied by NLRP3 inflammasome activation.

Experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), is mediated by myelin-specific autoreactive T-helper (Th) cells. Once Th cells are generated, their migration to the CNS is the next important step for EAE progression. Th cells infiltrate in the CNS by crossing the blood–brain barrier and mediate inflammatory responses, resulting in demyelination and neurodegeneration. Antigen-presenting cells (APCs), such as dendritic cells (DCs) and macrophages, also contribute to the progression of EAE by being recruited in the CNS. Together with CNS-resident APCs, recruited APCs restimulate CNS-infiltrated Th cells and eventually cause tissue damage together with Th cells in the CNS.

Factors that enhance immune cell migration play a critical role in EAE development. For example, mice deficient in CCR2, a major chemokine receptor, show severely compromised cell migration to spinal cords and are resistant to EAE (1). CCR2 antagonist and a neutralizing antibody for a CCR2 ligand, CCL2, suppress EAE progression (2, 3). EAE progression is also suppressed by treatment with the sphingosine 1-phosphate receptor 1 (S1PR1) agonist FTY720, which prevents T-cell egress from peripheral lymph nodes (4). In addition, blocking integrin α4, which promotes cell migration, suppresses progression of EAE and MS (5). FTY720 is now in clinical trials, and integrin α4 antibody (natalizumab) is currently used to treat MS patients. Targeting migration molecules can be quite effective in MS treatments.

The NLRP3 inflammasome senses pathogens and danger signals, such as bacteria, fungi, extracellular ATP, amyloid β, and uric acid. The NLRP3 inflammasome is a multiprotein complex, comprising NLR family, pyrin domain containing 3 (NLRP3), apoptosis-associated speck-like protein containing a carboxy-terminal CARD (ASC), and pro-caspase-1, and found in innate immune cells, such as macrophages and DCs. Active NLRP3 inflammasome processes pro–IL-1β and pro–IL-18 to produce mature IL-1β and IL-18, respectively. We and another group reported that mice lacking genes for Nlrp3 or Asc (Nlrp3^−/− and Asc^−/− mice) are resistant to the development of EAE (6, 7), suggesting the association of the NLRP3 inflammasome with EAE development. In MS plaques and/or cells from MS patients, the expression of caspase-1, IL-1β, and IL-18 is elevated (8–10), suggesting the involvement of the NLRP3 inflammasome in MS pathogenicity. However, the mechanism by which the NLRP3 inflammasome induces development of EAE and MS is poorly understood. In this study, we demonstrate that the NLRP3 inflammasome in APCs induces EAE development by enhancing chemokine-mediated immune cell recruitment in the CNS. In contrast, attenuated Th17 response in Nlrp3^−/− and Asc^−/− mice is not a determinative factor in their resistance against EAE. Therefore, inhibiting cell migration may be a good target if NLRP3 inflammasome activation induces progression of MS.

Results

Asc^−/− and Nlrp3^−/− Mice Were Resistant to EAE with Decreased Immune Cell Infiltration in the CNS.

We first observed that Asc^−/− and Nlrp3^−/− mice were resistant to EAE (Fig. 1A), as previously reported (6, 7). Because Asc^−/− and Nlrp3^−/− mice were equally resistant to EAE development (Fig. 1A), the NLRP3 inflammasome appeared to be required for disease progression. Asc^−/− mice showed little demyelination [Luxol fast blue (LFB) staining, Fig. 1B] and few infiltrating cells (H&E staining, Fig. 1B) in the spinal cord at the peak of disease (day 17). To enumerate cells infiltrated in the CNS, day 17 brains and spinal cords were harvested. Both Asc^−/− and Nlrp3^−/− mice displayed dramatically reduced numbers of total leukocytes and CD4⁺ T cells in spinal cords and brains (Fig. 1 C and D and Fig. S1 A–C). Furthermore, both Th17 and Th1 cells were almost completely absent in the CNS of Asc^−/− and Nlrp3^−/− mice (Fig. 1E and Fig. S1 C). Collectively, these data demonstrate that the NLRP3 inflammasome is required for EAE development, demyelination, and cell recruitment in the CNS.

Fig. 1.

Asc^−/− and Nlrp3^−/− mice are resistant to EAE. (A) EAE development. Representative data from three independent experiments are shown. Disease scores were presented as mean ± SEM for each group (n = 5). (B) LFB and H&E staining of spinal cord sections from WT and Asc^−/− mice at 17 d after EAE induction. Squares indicate representative regions shown at a high magnification on the right. Arrowheads indicate regions of demyelination. Representative data from three independent experiments are shown. (C and D) Numbers of total cells (C) and CD4⁺ T cells (D) obtained from spinal cords of WT, Asc^−/−, and Nlrp3^−/− mice at 17 d after EAE induction (n = 6–9). Horizontal lines denote mean values. (E) Intracellular staining of IL-17 and IFNγ and numbers of Th17 and Th1 cells in spinal cords of WT Asc and Nlrp3^−/− mice at 17 d after EAE induction (n = 6–8). *P < 0.05.

Reduced Th17 Response Does Not Account for EAE Resistance in Immunized Asc^−/− and Nlrp3^−/− Mice.

Inflammasome activation is required for maturation and secretion of IL-1β. We reported abundant levels of serum IL-1β in WT mice with EAE but not in immunized Asc^−/− and Nlrp3^−/− mice (7). Because IL-1β promotes Th17 cell generation (11, 12), we expected that deficiency of the NLRP3 inflammasome greatly attenuated Th17 cell population in our EAE model. However, Th17 cell numbers were reduced only about 50% in the draining lymph nodes (DLNs) of Asc^−/− and Nlrp3^−/− mice (Fig. 2A). Similar levels of partial reduction were seen in the proportion of Th17 cells and in concentrations of IL-17 in culture supernatants after in vitro stimulation of CD4⁺ T cells by Asc^−/− and Nlrp3^−/− DCs (Fig. 2 B and C). We found that numbers of splenic IL-17⁺ γδT cells were reduced about 50% in Asc^−/− and Nlrp3^−/− mice as well (Fig. S1D). Numbers of Th1 cell in the DLNs from immunized Asc^−/− and Nlrp3^−/− mice were also reduced to about half of those from WT mice (Fig. S1E), although proportions of Th1 cells were slightly increased in in vitro culture with Asc^−/− or Nlrp3^−/− DCs, (Fig. 2B). Thus, numbers of Th17, Th1, and IL-17⁺ γδT cells were reduced in Asc^−/− and Nlrp3^−/− mice, but only partially. Here, we speculated that such partial reduction may not fully account for the significant resistance against EAE in Asc^−/− and Nlrp3^−/− mice (Fig. 1A). To test whether cell numbers matter, we focused on the Th17 cell population, because Th17 cells were dominant over Th1 cells in DLNs (Fig. 2A and Fig. S1E). We obtained Th17 cells from immunized mice (WT, Asc^−/−, or Nlrp3^−/−) by cytokine capture beads and transferred the same number of Th17 cells to WT hosts. If a reduced number of IL-17⁺ cells is a reason for the EAE resistance in Asc^−/− and Nlrp3^−/− mice, recipients transferred with Asc^−/− or Nlrp3^−/− IL-17⁺ cells should develop EAE (Fig. 2D). However, EAE developed only in WT recipients transferred with Th17 cells but not with Asc^−/− and Nlrp3^−/− Th17 cells (Fig. 2E). This result suggests that the reduction of Th17 cells does not account for EAE resistance in immunized Asc^−/− and Nlrp3^−/− mice. Here, we evaluated the expression levels of IL-17 in Th17 cells isolated from WT, Asc^−/−, and Nlrp3^−/− mice, but expression levels of IL-17 were similar in Th17 cells from these mice (mean fluorescence intensity: WT, 292.7 ± 0.9; Asc^−/−, 305.3 ± 2.9; Nlrp3^−/−, 282.6 ± 0.2). Collectively, the quantity (i.e., the reduction) of Th17 population does not explain the resistance to EAE in Asc^−/− and Nlrp3^−/− mice, but the data here suggested that the quality of Th17 cells is altered in immunized Asc^−/− and Nlrp3^−/− mice.

Fig. 2.

Reduced Th17 response does not account for EAE resistance in Asc^−/− and Nlrp3^−/− mice. (A) Intracellular staining of IL-17 and IFNγ, and numbers of Th17 cells in DLNs at 9 d after EAE induction (n = 4–6). (B) In vitro Th17 cell generation. OT-2 CD4⁺ T cells were activated by splenic DCs from naïve mice with a Th17-polarizing condition. Flow cytometry plots show IL-17 and IFNγ intracellular staining in CD4⁺ T cells. Representative data from three independent experiments are shown. (C) IL-17 concentration in culture supernatants from experiments shown in B in triplicate wells. Representative data from three independent experiments are shown. (D) Schematic procedure for the experiment shown in E. IL-17⁺ cells were enriched by microbeads from WT, Asc^−/−, or Nlrp3^−/− mice at 9 d after immunization. IL-17⁺ cells (1 × 10⁶ cells per mouse) were adaptively transferred into Rag2^−/− mice. (E) Passive EAE induced by IL-17⁺ cell transfer. Disease scores were presented as mean ± SEM for each group (n = 5).

Immune Cell Accumulation in Peripheral Lymphoid Organs and Blood in Immunized Mice Deficient in the NLRP3 Inflammasome.

Decreased infiltration of CD4⁺ T cells in the CNS of Asc^−/− and Nlrp3^−/− mice may be caused by decreased cellularity in the periphery. To examine this possibility, we first evaluated cellularity in peripheral lymphoid organs and in blood on days 0, 9, and 17 after immunization. Although similar sizes of DLNs and spleens were observed in WT and Asc^−/− mice on days 0 and 9 (Fig. S2), DLNs and spleens were enlarged in Asc^−/− mice at the peak of EAE (day 17) (Fig. 3A, Top). Consistent with the enlargement of spleens and DLNs, numbers of total and myelin oligodendrocyte glycoprotein (MOG)-specific CD4⁺ T cells kept increasing in Asc^−/− mice by day 17 in DLNs and spleen, whereas these numbers started reducing in WT mice after day 9 (Fig. 3A and Fig. S3A). Numbers of these cells in blood also showed similar tendency, although the numbers in Asc^−/− mice did not keep increasing after day 9 (Fig. 3A). S1PR1 expressed in T cells directs their egress from lymph nodes into lymph and recirculation, but we found no alteration in expression of the S1pr1 gene and S1PR1 protein in total and MOG-specific CD4⁺ T cells from Asc^−/− mice (Fig. S3 B and C), suggesting no defect in T-cell egress from lymph nodes. These data demonstrated that NLRP3 inflammasome-deficient mice increased cellularity of circulating cells, including CD4⁺ T cells, at the disease peak.

Fig. 3.

Attenuated expression of genes encoding migration-related proteins impairs CD4⁺ T-cell migration in immunized Asc^−/− mice. (A Top) DLNs and spleens from WT and Asc^−/− mice at 17 d after EAE induction. (Middle and Bottom) Numbers of total cells (Middle) and CD4⁺ T cells (Bottom) in the DLNs, spleens, and peripheral blood in WT and Asc^−/− mice on the indicated days after EAE induction (n = 6–11). (B) Gene expression determined by qPCR in CD4⁺ T cells, Th17 cells, and Th1 cells (n = 4). (C) Naïve CD4⁺ T cells were stimulated with CD3/CD28 antibodies with or without rIL-1β (10 ng/mL) or rIL-18 (100 ng/mL) in tissue culture. Protein levels at 24 h after stimulation were determined by ELISA (secreted OPN) and FACS (CCR2 and CXCR6) (n = 4). Representative FACS data are presented in Fig. S4E. (D) CD4⁺ T-cell chemotaxis toward rCCL2 or rCXCL16 of indicated concentrations evaluated by a Transwell assay of triplicate wells. (B and D) Cells were obtained from spleens of WT or Asc^−/− mice at 9 d postimmunization. Representative data from two independent experiments are shown. *P < 0.05.

Normal T-Cell Proliferation and Cell Death in Immunized Asc^−/− and Nlrp3^−/− Mice.

It is not known whether Asc^−/− and Nlrp3^−/− mice have defects in generating and sustaining antigen-specific T cells during EAE development. Here, we found no significant difference between WT and Asc^−/− (or Nlrp3^−/−) mice in proportions and absolute numbers of MOG-specific CD4⁺ T cells on day 9 (Fig. S3A) and in in vivo T-cell proliferation (Fig. S3D). Asc^−/− and Nlrp3^−/− DCs also similarly proliferated MOG- and ovalbumin (OVA)-specific CD4⁺ T cells ex vivo (Fig. S3E). There was no significant difference in CD4⁺ T-cell necrosis and apoptosis as well (Fig. S3F). These results demonstrate that the proliferation and cell death of T cells is normal in the peripheral lymphoid organs of Asc^−/− and Nlrp3^−/− mice and suggest a defect in cell migration in these mice.

NLRP3 Inflammasome Increases Migration-Related Gene Expression and Chemotaxis of Th Cells.

To determine whether T cells activated in Asc^−/− and Nlrp3^−/− mice display an alteration in gene expression, we performed a microarray analysis using CD4⁺ T cells from DLNs and spleens of Asc^−/−, Nlrp3^−/−, and WT mice at 9 d after immunization. Day 9 is the time of EAE onset in WT mice; therefore, we considered that Th cell migration into the CNS is ongoing on day 9. A majority of genes with great expression reduction in Asc^−/− and Nlrp3^−/− mice turned out to encode chemokines, their receptors, and integrins. Migration-related genes that showed <50% expression in either DLNs or spleens of Asc^−/− and Nlrp3^−/− mice included Spp1, Ccr2, Ccl9, Ckap2, Ccl6, Ccr1, Ccl8, Vcam1, Cxcr6, Ccr6, and Ccr8 (Table S1). Expression levels of Spp1, Ccr2, Ccr1, Ccl9, and Cxcr6 genes were confirmed to be significantly lower in splenic CD4⁺ T cells in immunized Asc^−/− mice compared with those in immunized WT mice (Fig. 3B and Fig. S4A). We then examined gene expression in Th17 cells because the attenuated gene expression may simply be attributed to the reduction of the Th17 population size in total CD4⁺ T cells (Fig. 2A). IL-17 capture beads were used to isolate IL-17⁺ cells from spleens. Although Ccr1 and Ccl9 mRNA levels turned out to be similar between WT and Asc^−/− splenic Th17 cells (Fig. S4B), reduced Spp1, Ccr2, and Cxcr6 mRNA expression was still observed in splenic Th17 cells from immunized Asc^−/− mice (Fig. 3B). In addition, significant reduction of Spp1 and Cxcr6 mRNA expression was also observed in splenic Th1 cells from immunized Asc^−/− mice (Fig. 3B). These data suggest that Th17 and Th1 cells in immunized Asc^−/− mice have a different gene-expression pattern from that of WT Th17 and Th1 cells, indicating altered quality of Th cells.

We then sought to elucidate a molecular mechanism by which the NLRP3 inflammasome regulates migration-related genes in Th cells. NLRP3 inflammasome processes maturation of IL-1β and IL-18. We previously observed the elevated serum IL-1β and IL-18 production during EAE progression in WT mice (7). Therefore, we carried out ex vivo experiments to clarify the extent to which IL-1β and IL-18 up-regulate migration-related genes in WT CD4⁺ T cells. Naïve WT CD4⁺ T cells were stimulated with CD3/CD28 antibodies with or without recombinant (r)IL-1β or rIL-18. rIL-1β greatly enhanced mRNA expression of Spp1 and Cxcr6 and protein expression of osteopontin (OPN; Spp1 product) and CXCR6 (Fig. 3C and Fig. S4 C–E). rIL-18 also significantly enhanced mRNA expression of Ccr2 and Cxcr6 and protein expression of CCR2 and CXCR6 (Fig. 3C and Fig. S4 C–E).

Because expression of Ccr2 and Cxcr6 is decreased in CD4⁺ T cells from immunized Asc^−/− mice (Fig. 3B and Table S1), CD4⁺ T-cell chemotaxis toward CCL2 (CCR2 ligand) and CXCL16 (CXCR6 ligand), respectively, was evaluated by a Transwell assay. Significantly reduced chemotaxis toward both rCCL2 and rCXCL16 were observed in CD4⁺ T cells from immunized Asc^−/− mice (Fig. 3D), suggesting that attenuated gene expression of Ccr2 and Cxcr6 in CD4⁺ T cells from immunized Asc^−/− mice abated T-cell chemotaxis. These results confirm the critical involvement of ASC for Th cell migration by enhancing migration-related gene expression in the cells.

NLRP3 Inflammasome Increases the Expression of Genes Encoding Matching Chemokine/Receptor Pairs Between CD4⁺ T Cells and APCs.

We examined gene expression in CD4⁺ T cells, but the impaired cellular ability to migrate into the CNS was not limited to CD4⁺ T cells (Fig. 1C and Fig. S1A). Therefore, we asked whether macrophages and DCs attenuated expression of the genes that encodes matching chemokine/receptor counterparts of OPN, CCR2, and CXCR6. The α4β1 integrin is a receptor for OPN; CCL2, CCL7, and CCL8 are ligands of CCR2; and CXCL16 is a ligand of CXCR6. Significant reductions in mRNA levels of Itga4, Itgb1, Ccl7, Ccl8, and Cxcl16, but not Ccl2, were identified, particularly in macrophages from Asc^−/− mice at 9 d after immunization (Fig. 4 A and B and Fig. S4F). Furthermore, in tissue culture, we found that rIL-1β enhanced expression of Itga4 in DCs; of Ccl2, Ccl7 and Cxcl16 in macrophages and DCs; and of Ccl8 in macrophages (Fig. 4 C and D and Fig. S4G). rIL-18 enhanced Itga4, Ccl2, Ccl7, and Ccl8 in DCs and macrophages and Cxcl16 in macrophages (Fig. 4 C and D and Fig. S4G). Although the result left a possibility that some factors other than rIL-1β or rIL-18 also play a role in the induction of gene expression, IL-1β and IL-18 have a significant impact on up-regulating expression of a majority of genes that were examined.

Fig. 4.

DCs and macrophages from immunized Asc^−/− mice show attenuated expression of genes encoding chemokines or chemokine receptors. (A–D) Gene expression in macrophage (A and C) and DCs (B and D). Bone marrow-derived macrophages (C) and DCs (D) were treated with or without rIL-1β (10 ng/mL) or rIL-18 (100 ng/mL) and harvested at the indicated time points. mRNA levels were determined by qPCR (n = 4). *P < 0.05. (E) DC chemotaxis toward rOPN of indicated concentrations (n = 4). Integrin α4 antibody or control IgG was incubated with DCs for 1 h, and then DCs were plated in upper chamber of a Transwell. (A, B, and E) Cells were obtained from spleens of WT or Asc^−/− mice at 9 d after EAE induction. *P < 0.05 compared with WT DC data.

To evaluate a functional consequence of attenuated gene expression of Itga4 and Itgb1 in Asc^−/− DCs, we examined DC chemotaxis toward OPN by using DCs harvested from spleens in mice at day 9 postimmunization. DCs from immunized WT mice successfully migrated toward rOPN in an integrin α4-dependent manner (Fig. 4E and Fig. S4H). On the other hand, DCs from immunized Asc^−/− mice failed to migrate toward OPN (Fig. 4E and Fig. S4H). In summary, IL-1β and IL-18 up-regulate critical mediators of migration both in APCs and Th cells. The mediators included matching ligand/receptor combinations between T cells and APCs, such as CCR2 (T cells)/CCL7, CCL8, and CCL2 (APCs); CXCR6 (T cells)/CXCL16 (APCs); and OPN (T cells)/α4β1 integrin (APCs).

NLRP3 Inflammasome-Dependent Migration Defects Are Not T-Cell–Intrinsic.

The defects in T-cell migration seen in Asc^−/− and Nlrp3^−/− mice may be attributable to a loss of ASC or NLRP3 within the T cells themselves or to a loss of inflammasome activity within the APCs that stimulate T cells. WT CD4⁺ T cells expressed markedly less Nlrp3 and Casp1 mRNA than DCs do (Fig. 5A). Because particularly low expression levels of the Nlrp3 gene suggested little NLRP3 inflammasome activity in CD4⁺ T cells, we examined the possible impact of ASC expression on CD4⁺ T-cell encephalitogenicity. Naïve CD4⁺ T cells from WT or Asc^−/− mice were adoptively transferred into Rag2^−/− recipients (Asc^+/+), then the recipients were immunized to induce EAE (Fig. 5B). Rag2^−/− recipients transferred with naïve Asc^−/− CD4⁺ T cells developed EAE to the same extent as WT CD4⁺ T-cell–transferred Rag2^−/− recipients did (Fig. 5C). These results ruled out the involvement of the T-cell–intrinsic ASC and possible assembly of the NLRP3 inflammasome in CD4⁺ T cells during EAE development.

Fig. 5.

Presence of the NLRP3 inflammasome in APCs is sufficient to elicit T-cell migration. (A) Expression levels of genes encoding NLRP3 inflammasome components in DCs and CD4⁺ T cells were determined by qPCR. DCs and CD4⁺ T cells were obtained from spleen of WT mice at 9 d after immunization (n = 4). (B) Schematic procedure for the experiment shown in C. CD4⁺ T cells were isolated from spleens and lymph nodes of WT and Asc^−/− naïve donor mice and transferred (1 × 10⁶ cells) into Rag2^−/− recipients followed by MOG/complete Freund’s adjuvant (CFA) immunization. (C) EAE scores were presented as mean ± SEM for each group (n = 5). (D) Schematic procedure for the experiment shown in E. Naïve CD4⁺ 2D2 T cells were labeled with CFSE and transferred into WT, Asc^−/−, or Nlrp3^−/− mice that had been immunized at 2 d before the transfer. CD4⁺ T cells infiltrated into spinal cords and brains were enumerated at 4 d after the transfer. (E) Cell numbers of infiltrated CFSE-labeled CD4⁺ T cells into the spinal cord and brain (n = 5). *P < 0.05.

CD4⁺ T Cells Need to Be Primed by APCs That Express NLRP3 Inflammasome for Migration to the CNS.

On the basis of the above results, we hypothesized that Th cells need to be primed by APCs expressing the NLRP3 inflammasome to migrate to the CNS. To test this hypothesis, we examined in vivo activation of WT CD4⁺ T cells in Asc^−/− and Nlrp3^−/− hosts. Naïve 2D2 CD4⁺ T cells were labeled with carboxyfluorescein succinimidyl ester (CFSE) and adoptively transferred into WT, Asc^−/−, or Nlrp3^−/− hosts that had been preimmunized with MOG antigen (Fig. 5D). Migration of CFSE-labeled 2D2 T cells was evaluated at 4 d after the transfer. Although numbers of CFSE-labeled CD4⁺ T cells were similar in DLNs and spleens in all of the groups (Fig. S5), only WT hosts successfully recruited CFSE-labeled CD4⁺ T cells into the spinal cord and the brain (Fig. 5E). This result strongly suggests that the presence of the NLRP3 inflammasome in T-cell–priming APCs is essential for T-cell migration into the CNS.

I.v. Transfer of Th Cells Primed in Asc^−/− or Nlrp3^−/− Mice Does Not Induce EAE, but Direct Transfer to the CNS Does.

The findings above suggest that Th cells primed in Asc^−/− or Nlrp3^−/− mice are not encephalitogenic because of their inability to migrate into the CNS. To test T-cell migration, CD4⁺ T cells obtained from immunized WT, Asc^−/−, or Nlrp3^−/− mice at the time of EAE onset (day 9) were i.v. transferred to irradiated WT or Rag2^−/− recipients (Fig. 6A). CD4⁺ T cells obtained from immunized WT mice induced passive EAE and infiltrated into the CNS in irradiated WT recipients (Fig. 6 B and C), but CD4⁺ T cells from immunized Asc^−/− or Nlrp3^−/− mice failed to do so. Resistance to EAE by passive transfer of CD4⁺ T cells from immunized Asc^−/− and Nlrp3^−/− mice was also observed in Rag2^−/− recipient mice (Fig. 6D).

Fig. 6.

Bypassing the migration process to the CNS enables CD4⁺ T cells to induce EAE despite of priming in Asc^−/− or Nlrp3^−/− mice. (A) Schematic procedure for the experiments shown in B–G. (B–G) CD4⁺ T cells were obtained from spleens of WT, Asc^−/−, or Nlrp3^−/− mice at 9 d after immunization and transferred (3 × 10⁶ cells per mouse) into sublethally irradiated WT recipients (B and C) or Rag2^−/− recipients (D). CD4⁺ T cells (1 × 10⁶ cells per mouse) were also transferred directly into the brains (E) or spinal cords (F) of WT recipients by i.c.v. or i.th. injection, respectively, or by the combination of both i.c.v. and i.th. injections (G). G also includes negative controls with splenic CD4⁺ T cells from naïve WT (△), Asc^−/− (▲) or Nlrp3^−/− (◆) mice transferred (1 × 10⁶ cells per mouse) to recipients (no EAE developed). (B and D–G) EAE scores were presented as mean ± SEM for each group (n = 5). (C) Numbers of CD4⁺ T cells in spinal cords on day 44 after CD4⁺ T-cell transfer (n = 5). *P < 0.05.

Next, we directly transferred CD4⁺ T cells into the brain or spinal cord by i.c.v. or intrathecal (i.th.) injection, respectively, to bypass the cell migration process. CD4⁺ T cells from immunized WT, Asc^−/−, or Nlrp3^−/− mice developed similar levels of EAE (Fig. 6 E and F). When CD4⁺ T cells were transferred into both the brain and spinal cord, EAE was more severe than with i.c.v. or i.th. injection alone, and CD4⁺ T cells from immunized Asc^−/− or Nlrp3^−/− mice again induced similar levels of EAE (Fig. 6G), as well as demyelination in spinal cord (Fig. S6), as WT CD4⁺ T cells did. Congruent with EAE scores, demyelination by i.c.v. and i.th. transfer of CD4⁺ T cells was milder than that in immunized WT mice or Rag2^−/− with i.v. transfer of CD4⁺ T cells (Fig. S6). These data suggest that the cell migration is indeed the determinative factor for NLRP3 inflammasome-mediated EAE development.

Discussion

This study and previous ones (6, 7, 13) showed that Nlrp3^−/− mice are resistant to the development of EAE, suggesting the association of the NLRP3 inflammasome with EAE development. We also showed that Asc^−/− mice were resistant to EAE as Nlrp3^−/− mice. Because it is not clear how the NLRP3 inflammasome enhances EAE, we sought to elucidate the mechanism in this study. Currently, the attenuated Th17 cell responses are suggested to be a major underlying mechanism for the resistance of knockout mice to EAE (13, 14). Indeed, a number of studies demonstrated the critical role of Th17 responses in EAE development and the promotion of Th17 cell generation by IL-1β. Based on the reduced Th17 population in immunized Asc^−/− and Nlrp3^−/− mice, it is quite reasonable to consider that Th17 mediates the impact of the NLRP3 inflammasome on EAE development. However, we found that the reduction of the Th17 population does not account for the resistance to EAE in Asc^−/− and Nlrp3^−/− mice. Instead, the NLRP3 inflammasome is required for Th17 cells to enhance their migration ability to the CNS. Not only Th17 cells but APCs and Th1 cells were also found to enhance chemotaxis by the NLRP3 inflammasome.

Our data from microarray and quantitative PCR (qPCR) analyses showed that Th cells (and the Th17 cell population alone) from immunized Asc^−/− and Nlrp3^−/− mice showed less expression of several migration-related molecules, such as Spp1, Ccr2, and Cxcr6. Spp1 encodes OPN. As a ligand of various integrins, including the α4β1 integrin, OPN plays a role in attracting immune cells (15). In addition to high expression of OPN in MS lesions (16), Spp1^−/− mice develop milder EAE than WT mice did (16–19). The blockade of α4β1 integrin was also shown to reduce relapse rates in relapsing–remitting MS patients and to delay progression of the disease (20). CCL2 is one of the CCR2 ligands. Ccr2^−/− and Ccl2^−/− mice both show reduced mononuclear cell infiltrate in the CNS with decreased susceptibility to EAE (1, 21). Previous studies showed that CCR2 expression in circulating CD4⁺ T cells is significantly elevated during MS relapse (22, 23). CXCR6 is required for neuroinflammation by immune cell infiltration in cortical injury sites (24). Although Cxcr6^−/− mice develop EAE to a similar extent as WT mice, antibodies against CXCL16 are known to reduce EAE severity (24, 25). Th1 cell trafficking is reported to be independent of the α4 integrin (26), but for the optimal expression of chemotactic molecules, such as Spp1 and Cxcr6, the NLRP3 inflammasome is still needed. Indeed, Th1 cells were not detected in the CNS of immunized Asc^−/− and Nlrp3^−/− mice. Therefore, despite the different migration machinery of Th1 cells from that of Th17 cells, CNS infiltration of both Th subsets is greatly compromised in immunized Asc^−/− and Nlrp3^−/− mice. In summary, the NLRP3 inflammasome up-regulates expression of migration-enhancing molecules (summarized in Fig. S7), which are involved in development of EAE and probably in MS as well.

Our study further showed that T cells need to be primed in NLRP3 inflammasome-sufficient mice to migrate into the CNS and induce EAE (Fig. S7), although the full chain of events is probably intricately regulated, and we do not rule out the involvement of factors other than the NLRP3 inflammasome. Among the impacts of the NLRP3 inflammasome on cell migration, we demonstrated the involvement of IL-1β and IL-18. It is of note that inflammasomes induce pyroptotic cell death in addition to maturation of the cytokines. For EAE induction, the involvement of cellular contents released by NLRP3 inflammasome-mediated pyroptotic cell death into the microenvironment is possible. On the other hand, a recent study showed regulation of actin polymerization by ASC (27), which may also contribute to cell migration. However, our results here showed that the defective phenotype of cell migration in immunized Nlrp3^−/− mice is very similar to that of Asc^−/− mice, i.e., not specific to ASC. Therefore, at least in this EAE model, ASC-specific impairment of actin polymerization does not seem to play a major role in cell migration. In addition, we have shown similar levels of cellularity in splenocytes and lymph nodes among naïve WT, Asc^−/−, and Nlrp3^−/− mice. In contrast, Ippagunta et al. (27) showed greatly reduced cellularity in T cells, B cells, and CD11c⁺ cells in Asc^−/− mice. The reason for the discrepancy is currently unknown.

We observed that direct CD4⁺ T-cell injection into the CNS induced much milder EAE and demyelination compared with EAE induced by CD4⁺ T-cell i.v. injection. As shown in this study and an article by another group (28), direct T-cell injection into the CNS may not be an aggressive approach to induce EAE, as it may sound. Severe EAE is developed by i.v. CD4⁺ T-cell transfer but not by direct CD4⁺ T-cell transfer to the CNS, because i.v. injection allows time and space for transferred T cells to proliferate in the periphery before they infiltrate into the CNS. In addition, it is widely known that APCs are recruited together from the periphery to the CNS in passive EAE induced by i.v. T-cell transfer. APCs infiltrated in the CNS restimulate CNS-infiltrated autoreactive T cells, further contributing to the development of EAE. In the case of direct T-cell transfer to the CNS, no inflammatory cell expansion in the periphery is expected as well as extra inflammatory cell recruitment from the periphery. In addition, artificial T-cell injection to the CNS is technically not as efficient as natural T-cell recruitment into the CNS. A recent article showed that autoreactive T cells access the CNS via the fifth lumber spinal cord to induce EAE (29). This extremely defined route may make artificial cell injections inefficient because of the requirement of transferred T cells to be precisely targeted to the defined route for effective elicitation of their encephalitogenicity.

There are a number of reports that strongly suggest the involvement of inflammasomes in MS development. It is possible that activation of the NLRP3 inflammasome induces inflammatory cell recruitment into the CNS. Our study suggests a strong connection between the NLRP3 inflammasome and immune cell migration through induction of chemokines and their receptors. As currently applied in clinical interventions of MS, targeting molecules that enhance immune cell migration appears to be an effective approach in treating MS accompanied with NLRP3 inflammasome activation.

Materials and Methods

Animals.

Male mice of the C57BL/6 background were used in this study. The Asc^−/− and Nlrp3^−/− mice were a gift from Genentech and were rederived in our facility. The 2D2 and OT-2 T-cell receptor (TCR) transgenic (Tg) mice were purchased from The Jackson Laboratory. The mice were kept in a barrier facility. This study was approved by the Duke University Institutional Animal Care and Use Committee. EAE induction was performed as previously described (18).

Adoptive Transfer of CD4⁺ Th Cells and Th17 Cells.

CD4⁺ T cells, IL-17⁺, and IFNγ⁺ cells were isolated from spleens and DLNs of WT, Asc^−/−, or Nlrp3^−/− mice at 9 d after EAE induction by positive selection by using CD4 microbeads or IL-17- or IFNγ-capture microbeads (Miltenyi Biotec). Isolated T cells were adoptively transferred by i.v. injection to Rag2^−/− recipient mice or sublethally irradiated WT mice (irradiation was performed 24 h before T-cell transfer). Mice were also i.p. injected with pertussis toxin on days 0 and 2. In some experiments, isolated CD4⁺ Th cells were adoptively transferred by i.th. and/or i.c.v. injection to WT mice with i.p. injection of pertussis toxin on day −4, −2, 0, and 2 (where day 0 is T-cell transfer).

Statistical Analysis.

Statistical analysis was performed with Student’s t tests. The criterion of significance was set as P < 0.05. All results are expressed as mean ± SEM.

All other methods and further details are provided in SI Materials and Methods. Primer sequences are shown in Table S2.