NLRP3 inflammasome induces chemotactic immune cellmigration to the CNS in experimentalautoimmune encephalomyelitisMakoto Inouea, Kristi L. Williamsb,c, Michael D. Gunnb, and Mari L. Shinoharaa,d,1

Departments of aImmunology, bMedicine/Cardiology, and dMolecular Genetics and Microbiology, and cSchool of Nursing, Duke University Medical Center,Durham, NC 27710

Edited* by Harvey Cantor, Dana-Farber Cancer Institute, Boston, MA, and approved May 16, 2012 (received for review February 1, 2012)

The NLRP3 inflammasome is a multiprotein complex consisting ofthree kinds of proteins, NLRP3, ASC, and pro-caspase-1, and playsa role in sensing pathogens and danger signals in the innate im-mune system. The NLRP3 inflammasome is thought to be involvedin the development of experimental autoimmune encephalomyeli-tis (EAE), an animal model of multiple sclerosis (MS). However, themechanism by which the NLRP3 inflammasome induces EAE is notclear. In this study, we found that the NLRP3 inflammasomeplayed a critical role in inducing T-helper cell migration into theCNS. To gain migratory ability, CD4+ T cells need to be primed byNLRP3 inflammasome-sufficient antigen-presenting cells to up-regulate chemotaxis-related proteins, such as osteopontin, CCR2,and CXCR6. In the presence of the NLRP3 inflammasome, dendriticcells and macrophages also induce chemotactic ability and up-reg-ulate chemotaxis-related proteins, such as α4β1 integrin, CCL7,CCL8, and CXCL16. On the other hand, reduced Th17 cell popula-tion size in immunized Nlrp3−/− and Asc−/− mice is not a determi-native factor for their resistance to EAE. As currently applied inclinical interventions of MS, targeting immune cell migration mol-ecules may be an effective approach in treating MS accompaniedby NLRP3 inflammasome activation.

neuroinflammation | passive experimental autoimmune encephalomyelitis |demyelination | intrathecal injection | intracerebroventricular injection

Experimental autoimmune encephalomyelitis (EAE), an ani-mal model of multiple sclerosis (MS), is mediated by myelin-

specific autoreactive T-helper (Th) cells. Once Th cells aregenerated, their migration to the CNS is the next important stepfor EAE progression. Th cells infiltrate in the CNS by crossingthe blood–brain barrier and mediate inflammatory responses,resulting in demyelination and neurodegeneration. Antigen-presenting cells (APCs), such as dendritic cells (DCs) and mac-rophages, also contribute to the progression of EAE by beingrecruited in the CNS. Together with CNS-resident APCs,recruited APCs restimulate CNS-infiltrated Th cells and even-tually 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 cellmigration to spinal cords and are resistant to EAE (1). CCR2antagonist and a neutralizing antibody for a CCR2 ligand, CCL2,suppress EAE progression (2, 3). EAE progression is also sup-pressed by treatment with the sphingosine 1-phosphate receptor1 (S1PR1) agonist FTY720, which prevents T-cell egress fromperipheral lymph nodes (4). In addition, blocking integrin α4,which promotes cell migration, suppresses progression of EAE andMS (5). FTY720 is now in clinical trials, and integrin α4 antibody(natalizumab) is currently used to treat MS patients. Targetingmigration molecules can be quite effective in MS treatments.The NLRP3 inflammasome senses pathogens and danger

signals, such as bacteria, fungi, extracellular ATP, amyloid β, anduric 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 innateimmune cells, such as macrophages and DCs. Active NLRP3inflammasome processes pro–IL-1β and pro–IL-18 to producemature IL-1β and IL-18, respectively. We and another groupreported that mice lacking genes for Nlrp3 or Asc (Nlrp3−/− andAsc−/− mice) are resistant to the development of EAE (6, 7),suggesting the association of the NLRP3 inflammasome withEAE development. In MS plaques and/or cells fromMS patients,the expression of caspase-1, IL-1β, and IL-18 is elevated (8–10),suggesting the involvement of the NLRP3 inflammasome in MSpathogenicity. However, the mechanism by which the NLRP3inflammasome induces development of EAE and MS is poorlyunderstood. In this study, we demonstrate that the NLRP3inflammasome in APCs induces EAE development by enhancingchemokine-mediated immune cell recruitment in the CNS. Incontrast, attenuated Th17 response in Nlrp3−/− and Asc−/− miceis not a determinative factor in their resistance against EAE.Therefore, inhibiting cell migration may be a good target ifNLRP3 inflammasome activation induces progression of MS.

ResultsAsc−/− and Nlrp3−/− Mice Were Resistant to EAE with DecreasedImmune Cell Infiltration in the CNS. We first observed that Asc−/−

and Nlrp3−/− mice were resistant to EAE (Fig. 1A), as previouslyreported (6, 7). Because Asc−/− and Nlrp3−/− mice were equallyresistant to EAE development (Fig. 1A), the NLRP3 inflam-masome 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 thespinal cord at the peak of disease (day 17). To enumerate cellsinfiltrated in the CNS, day 17 brains and spinal cords wereharvested. Both Asc−/− and Nlrp3−/− mice displayed dramaticallyreduced numbers of total leukocytes and CD4+ T cells in spinalcords and brains (Fig. 1 C and D and Fig. S1 A–C). Furthermore,both Th17 and Th1 cells were almost completely absent in theCNS of Asc−/− and Nlrp3−/− mice (Fig. 1E and Fig. S1 C). Col-lectively, these data demonstrate that the NLRP3 inflammasomeis required for EAE development, demyelination, and cell re-cruitment in the CNS.

Reduced Th17 Response Does Not Account for EAE Resistance inImmunized Asc−/− and Nlrp3−/− Mice. Inflammasome activation isrequired for maturation and secretion of IL-1β. We reported

Author contributions: M.I. and M.L.S. designed research; M.I. performed research; K.L.W.contributed new reagents/analytic tools; M.I., M.D.G., and M.L.S. analyzed data; and M.I.and M.L.S. wrote the paper.

The authors declare no conflict of interest.

*This Direct Submission article had a prearranged editor.1To whom correspondence should be addressed. E-mail: mari.shinohara@duke.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1201836109/-/DCSupplemental.

10480–10485 | PNAS | June 26, 2012 | vol. 109 | no. 26 www.pnas.org/cgi/doi/10.1073/pnas.1201836109

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abundant levels of serum IL-1β in WT mice with EAE but not inimmunized Asc−/− and Nlrp3−/− mice (7). Because IL-1β pro-motes Th17 cell generation (11, 12), we expected that deficiencyof the NLRP3 inflammasome greatly attenuated Th17 cell pop-ulation in our EAE model. However, Th17 cell numbers werereduced only about 50% in the draining lymph nodes (DLNs) ofAsc−/− and Nlrp3−/− mice (Fig. 2A). Similar levels of partial re-duction were seen in the proportion of Th17 cells and in con-centrations of IL-17 in culture supernatants after in vitrostimulation of CD4+ T cells by Asc−/− and Nlrp3−/− DCs (Fig. 2 Band C). We found that numbers of splenic IL-17+ γδT cells werereduced 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 fromWT mice (Fig. S1E), although proportions of Th1 cells wereslightly increased in in vitro culture with Asc−/− or Nlrp3−/− DCs,(Fig. 2B). Thus, numbers of Th17, Th1, and IL-17+ γδT cellswere reduced in Asc−/− and Nlrp3−/− mice, but only partially.Here, we speculated that such partial reduction may not fullyaccount for the significant resistance against EAE in Asc−/− andNlrp3−/− mice (Fig. 1A). To test whether cell numbers matter, wefocused on the Th17 cell population, because Th17 cells weredominant over Th1 cells in DLNs (Fig. 2A and Fig. S1E). Weobtained Th17 cells from immunized mice (WT, Asc−/−, orNlrp3−/−) by cytokine capture beads and transferred the samenumber 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 developedonly in WT recipients transferred with Th17 cells but not withAsc−/− and Nlrp3−/− Th17 cells (Fig. 2E). This result suggests thatthe reduction of Th17 cells does not account for EAE resistancein immunized Asc−/− and Nlrp3−/− mice. Here, we evaluated theexpression levels of IL-17 in Th17 cells isolated fromWT, Asc−/−,and Nlrp3−/− mice, but expression levels of IL-17 were similar inTh17 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 populationdoes not explain the resistance to EAE in Asc−/− and Nlrp3−/−

mice, but the data here suggested that the quality of Th17 cells isaltered in immunized Asc−/− and Nlrp3−/− mice.

Immune Cell Accumulation in Peripheral Lymphoid Organs and Bloodin Immunized Mice Deficient in the NLRP3 Inflammasome. Decreasedinfiltration of CD4+ T cells in the CNS of Asc−/− and Nlrp3−/−

mice may be caused by decreased cellularity in the periphery. Toexamine this possibility, we first evaluated cellularity in periph-eral lymphoid organs and in blood on days 0, 9, and 17 afterimmunization. Although similar sizes of DLNs and spleens wereobserved in WT and Asc−/− mice on days 0 and 9 (Fig. S2), DLNsand spleens were enlarged in Asc−/− mice at the peak of EAE(day 17) (Fig. 3A, Top). Consistent with the enlargement ofspleens and DLNs, numbers of total and myelin oligodendrocyteglycoprotein (MOG)-specific CD4+ T cells kept increasing inAsc−/− mice by day 17 in DLNs and spleen, whereas thesenumbers started reducing in WT mice after day 9 (Fig. 3A andFig. S3A). Numbers of these cells in blood also showed similartendency, although the numbers in Asc−/− mice did not keepincreasing after day 9 (Fig. 3A). S1PR1 expressed in T cellsdirects their egress from lymph nodes into lymph and recircu-lation, but we found no alteration in expression of the S1pr1 geneand S1PR1 protein in total and MOG-specific CD4+ T cells fromAsc−/− mice (Fig. S3 B and C), suggesting no defect in T-cellegress from lymph nodes. These data demonstrated that NLRP3inflammasome-deficient mice increased cellularity of circulatingcells, including CD4+ T cells, at the disease peak.

Normal T-Cell Proliferation and Cell Death in Immunized Asc−/− andNlrp3−/− Mice. It is not known whether Asc−/− and Nlrp3−/− micehave defects in generating and sustaining antigen-specific T cellsduring EAE development. Here, we found no significant

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Fig. 1. Asc−/− and Nlrp3−/− mice are resistant to EAE. (A) EAE development.Representative data from three independent experiments are shown. Dis-ease scores were presented as mean ± SEM for each group (n = 5). (B) LFBand H&E staining of spinal cord sections from WT and Asc−/− mice at 17 dafter EAE induction. Squares indicate representative regions shown at a highmagnification on the right. Arrowheads indicate regions of demyelination.Representative data from three independent experiments are shown. (C andD) Numbers of total cells (C) and CD4+ T cells (D) obtained from spinal cordsof WT, Asc−/−, and Nlrp3−/− mice at 17 d after EAE induction (n = 6–9).Horizontal lines denote mean values. (E) Intracellular staining of IL-17and IFNγ and numbers of Th17 and Th1 cells in spinal cords of WT Asc andNlrp3−/− mice at 17 d after EAE induction (n = 6–8). *P < 0.05.

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and Nlrp3−/− mice. (A) Intracellular staining of IL-17 and IFNγ, and numbersof Th17 cells in DLNs at 9 d after EAE induction (n = 4–6). (B) In vitro Th17 cellgeneration. OT-2 CD4+ T cells were activated by splenic DCs from naïve micewith a Th17-polarizing condition. Flow cytometry plots show IL-17 and IFNγintracellular staining in CD4+ T cells. Representative data from three in-dependent experiments are shown. (C) IL-17 concentration in culturesupernatants from experiments shown in B in triplicate wells. Representativedata from three independent experiments are shown. (D) Schematic pro-cedure for the experiment shown in E. IL-17+ cells were enriched bymicrobeads 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 werepresented as mean ± SEM for each group (n = 5).

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difference between WT and Asc−/− (or Nlrp3−/−) mice in pro-portions and absolute numbers of MOG-specific CD4+ T cellson day 9 (Fig. S3A) and in in vivo T-cell proliferation (Fig. S3D).Asc−/− and Nlrp3−/− DCs also similarly proliferated MOG- andovalbumin (OVA)-specific CD4+ T cells ex vivo (Fig. S3E).There was no significant difference in CD4+ T-cell necrosis andapoptosis as well (Fig. S3F). These results demonstrate that theproliferation and cell death of T cells is normal in the peripherallymphoid organs of Asc−/− and Nlrp3−/− mice and suggest a de-fect in cell migration in these mice.

NLRP3 Inflammasome Increases Migration-Related Gene Expressionand Chemotaxis of Th Cells. To determine whether T cells activatedin Asc−/− and Nlrp3−/− mice display an alteration in gene ex-pression, we performed a microarray analysis using CD4+ T cellsfrom DLNs and spleens of Asc−/−, Nlrp3−/−, and WT mice at 9 dafter immunization. Day 9 is the time of EAE onset in WT mice;therefore, we considered that Th cell migration into the CNS isongoing on day 9. A majority of genes with great expressionreduction in Asc−/− and Nlrp3−/− mice turned out to encodechemokines, their receptors, and integrins. Migration-relatedgenes that showed <50% expression in either DLNs or spleens ofAsc−/− and Nlrp3−/− mice included Spp1, Ccr2, Ccl9, Ckap2, Ccl6,Ccr1, Ccl8, Vcam1, Cxcr6, Ccr6, and Ccr8 (Table S1). Expressionlevels of Spp1, Ccr2, Ccr1, Ccl9, and Cxcr6 genes were confirmedto be significantly lower in splenic CD4+ T cells in immunizedAsc−/− 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 at-tributed to the reduction of the Th17 population size in totalCD4+ T cells (Fig. 2A). IL-17 capture beads were used to isolateIL-17+ cells from spleens. Although Ccr1 and Ccl9 mRNA levelsturned out to be similar between WT and Asc−/− splenic Th17cells (Fig. S4B), reduced Spp1, Ccr2, and Cxcr6 mRNA expres-sion was still observed in splenic Th17 cells from immunizedAsc−/− mice (Fig. 3B). In addition, significant reduction of Spp1and Cxcr6 mRNA expression was also observed in splenic Th1cells from immunized Asc−/− mice (Fig. 3B). These data suggestthat Th17 and Th1 cells in immunized Asc−/− mice have a dif-ferent gene-expression pattern from that of WT Th17 and Th1cells, indicating altered quality of Th cells.We then sought to elucidate a molecular mechanism by which

the NLRP3 inflammasome regulates migration-related genes inTh cells. NLRP3 inflammasome processes maturation of IL-1βand IL-18. We previously observed the elevated serum IL-1βand IL-18 production during EAE progression in WT mice (7).Therefore, we carried out ex vivo experiments to clarify the ex-tent to which IL-1β and IL-18 up-regulate migration-relatedgenes in WT CD4+ T cells. Naïve WT CD4+ T cells were stim-ulated with CD3/CD28 antibodies with or without recombinant(r)IL-1β or rIL-18. rIL-1β greatly enhanced mRNA expressionof Spp1 and Cxcr6 and protein expression of osteopontin (OPN;Spp1 product) and CXCR6 (Fig. 3C and Fig. S4 C–E). rIL-18also significantly enhanced mRNA expression of Ccr2 andCxcr6 and protein expression of CCR2 and CXCR6 (Fig. 3Cand 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) andCXCL16 (CXCR6 ligand), respectively, was evaluated bya Transwell assay. Significantly reduced chemotaxis toward bothrCCL2 and rCXCL16 were observed in CD4+ T cells from im-munized Asc−/− mice (Fig. 3D), suggesting that attenuated geneexpression of Ccr2 and Cxcr6 in CD4+ T cells from immunizedAsc−/− mice abated T-cell chemotaxis. These results confirm thecritical involvement of ASC for Th cell migration by enhancingmigration-related gene expression in the cells.

NLRP3 Inflammasome Increases the Expression of Genes EncodingMatching Chemokine/Receptor Pairs Between CD4+ T Cells and APCs.We examined gene expression in CD4+ T cells, but the impairedcellular ability to migrate into the CNS was not limited to CD4+

T cells (Fig. 1C and Fig. S1A). Therefore, we asked whethermacrophages and DCs attenuated expression of the genes thatencodes 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 isa ligand of CXCR6. Significant reductions in mRNA levels ofItga4, Itgb1, Ccl7, Ccl8, and Cxcl16, but not Ccl2, were identified,particularly in macrophages from Asc−/− mice at 9 d after im-munization (Fig. 4 A and B and Fig. S4F). Furthermore, in tissueculture, we found that rIL-1β enhanced expression of Itga4 inDCs; of Ccl2, Ccl7 and Cxcl16 in macrophages and DCs; and ofCcl8 in macrophages (Fig. 4 C and D and Fig. S4G). rIL-18enhanced Itga4, Ccl2, Ccl7, and Ccl8 in DCs and macrophagesand Cxcl16 in macrophages (Fig. 4 C and D and Fig. S4G). Al-though the result left a possibility that some factors other thanrIL-1β or rIL-18 also play a role in the induction of gene ex-pression, IL-1β and IL-18 have a significant impact on up-regu-lating expression of a majority of genes that were examined.To evaluate a functional consequence of attenuated gene ex-

pression of Itga4 and Itgb1 in Asc−/− DCs, we examined DCchemotaxis toward OPN by using DCs harvested from spleens inmice at day 9 postimmunization. DCs from immunized WT micesuccessfully migrated toward rOPN in an integrin α4-dependentmanner (Fig. 4E and Fig. S4H). On the other hand, DCs from

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immunized Asc−/− mice failed to migrate toward OPN (Fig. 4Eand Fig. S4H). In summary, IL-1β and IL-18 up-regulate criticalmediators of migration both in APCs and Th cells. The media-tors included matching ligand/receptor combinations betweenT cells and APCs, such as CCR2 (T cells)/CCL7, CCL8, andCCL2 (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−/− andNlrp3−/− mice may be attributable to a loss of ASC or NLRP3within the T cells themselves or to a loss of inflammasome ac-tivity within the APCs that stimulate T cells. WT CD4+ T cellsexpressed markedly less Nlrp3 and Casp1 mRNA than DCs do(Fig. 5A). Because particularly low expression levels of the Nlrp3gene suggested little NLRP3 inflammasome activity in CD4+

T cells, we examined the possible impact of ASC expression onCD4+ T-cell encephalitogenicity. Naïve CD4+ T cells from WTor Asc−/− mice were adoptively transferred into Rag2−/− recipi-ents (Asc+/+), then the recipients were immunized to induceEAE (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 resultsruled out the involvement of the T-cell–intrinsic ASC and pos-sible assembly of the NLRP3 inflammasome in CD4+ T cellsduring EAE development.

CD4+ T Cells Need to Be Primed by APCs That Express NLRP3 In-flammasome for Migration to the CNS. On the basis of the aboveresults, we hypothesized that Th cells need to be primed by APCsexpressing the NLRP3 inflammasome to migrate to the CNS. Totest this hypothesis, we examined in vivo activation of WT CD4+

T cells in Asc−/− and Nlrp3−/− hosts. Naïve 2D2 CD4+ T cellswere labeled with carboxyfluorescein succinimidyl ester (CFSE)

and adoptively transferred into WT, Asc−/−, or Nlrp3−/− hosts thathad been preimmunized with MOG antigen (Fig. 5D). Migrationof CFSE-labeled 2D2 T cells was evaluated at 4 d after thetransfer. Although numbers of CFSE-labeled CD4+ T cells weresimilar in DLNs and spleens in all of the groups (Fig. S5), onlyWT hosts successfully recruited CFSE-labeled CD4+ T cells intothe spinal cord and the brain (Fig. 5E). This result strongly sug-gests 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 NotInduce EAE, but Direct Transfer to the CNS Does. The findings abovesuggest that Th cells primed in Asc−/− or Nlrp3−/− mice are notencephalitogenic because of their inability to migrate into the CNS.To test T-cell migration, CD4+ T cells obtained from immunizedWT, 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 inducedpassive EAE and infiltrated into the CNS in irradiated WTrecipients (Fig. 6 B and C), but CD4+ T cells from immunizedAsc−/− or Nlrp3−/− mice failed to do so. Resistance to EAE bypassive transfer of CD4+ T cells from immunized Asc−/− andNlrp3−/− mice was also observed in Rag2−/− recipient mice(Fig. 6D).Next, we directly transferred CD4+ T cells into the brain or

spinal cord by i.c.v. or intrathecal (i.th.) injection, respectively, tobypass the cell migration process. CD4+ T cells from immunizedWT, Asc−/−, or Nlrp3−/− mice developed similar levels of EAE(Fig. 6 E and F). When CD4+ T cells were transferred into boththe 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 im-munized WT mice or Rag2−/− with i.v. transfer of CD4+ T cells

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Fig. 4. DCs and macrophages from immunized Asc−/− mice show attenu-ated expression of genes encoding chemokines or chemokine receptors. (A–D) Gene expression in macrophage (A and C) and DCs (B and D). Bonemarrow-derived macrophages (C) and DCs (D) were treated with or withoutrIL-1β (10 ng/mL) or rIL-18 (100 ng/mL) and harvested at the indicated timepoints. mRNA levels were determined by qPCR (n = 4). *P < 0.05. (E) DCchemotaxis toward rOPN of indicated concentrations (n = 4). Integrin α4antibody or control IgG was incubated with DCs for 1 h, and then DCs wereplated in upper chamber of a Transwell. (A, B, and E) Cells were obtainedfrom spleens of WT or Asc−/− mice at 9 d after EAE induction. *P < 0.05compared with WT DC data.

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(Fig. S6). These data suggest that the cell migration is indeedthe determinative factor for NLRP3 inflammasome-mediatedEAE development.

DiscussionThis study and previous ones (6, 7, 13) showed that Nlrp3−/− miceare resistant to the development of EAE, suggesting the asso-ciation of the NLRP3 inflammasome with EAE development.We also showed that Asc−/− mice were resistant to EAE asNlrp3−/− mice. Because it is not clear how the NLRP3 inflam-masome enhances EAE, we sought to elucidate the mechanismin this study. Currently, the attenuated Th17 cell responses aresuggested to be a major underlying mechanism for the resistanceof knockout mice to EAE (13, 14). Indeed, a number of studiesdemonstrated the critical role of Th17 responses in EAE de-velopment and the promotion of Th17 cell generation by IL-1β.Based on the reduced Th17 population in immunized Asc−/− andNlrp3−/− mice, it is quite reasonable to consider that Th17mediates the impact of the NLRP3 inflammasome on EAE de-velopment. However, we found that the reduction of the Th17population does not account for the resistance to EAE in Asc−/−

and Nlrp3−/− mice. Instead, the NLRP3 inflammasome is re-quired for Th17 cells to enhance their migration ability to theCNS. Not only Th17 cells but APCs and Th1 cells were alsofound to enhance chemotaxis by the NLRP3 inflammasome.Our data from microarray and quantitative PCR (qPCR)

analyses showed that Th cells (and the Th17 cell populationalone) from immunized Asc−/− and Nlrp3−/− mice showed lessexpression 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 inattracting immune cells (15). In addition to high expression ofOPN in MS lesions (16), Spp1−/− mice develop milder EAE thanWT mice did (16–19). The blockade of α4β1 integrin was alsoshown to reduce relapse rates in relapsing–remitting MS patientsand to delay progression of the disease (20). CCL2 is one of theCCR2 ligands. Ccr2−/− and Ccl2−/− mice both show reducedmononuclear cell infiltrate in the CNS with decreased suscepti-bility to EAE (1, 21). Previous studies showed that CCR2 ex-pression in circulating CD4+ T cells is significantly elevatedduring MS relapse (22, 23). CXCR6 is required for neuro-inflammation by immune cell infiltration in cortical injury sites(24). Although Cxcr6−/− mice develop EAE to a similar extent asWT mice, antibodies against CXCL16 are known to reduce EAEseverity (24, 25). Th1 cell trafficking is reported to be in-dependent of the α4 integrin (26), but for the optimal expressionof chemotactic molecules, such as Spp1 and Cxcr6, the NLRP3inflammasome is still needed. Indeed, Th1 cells were notdetected in the CNS of immunized Asc−/− and Nlrp3−/− mice.Therefore, despite the different migration machinery of Th1cells from that of Th17 cells, CNS infiltration of both Th subsetsis greatly compromised in immunized Asc−/− and Nlrp3−/− mice.In summary, the NLRP3 inflammasome up-regulates expressionof migration-enhancing molecules (summarized in Fig. S7),which are involved in development of EAE and probably in MSas well.Our study further showed that T cells need to be primed in

NLRP3 inflammasome-sufficient mice to migrate into the CNSand induce EAE (Fig. S7), although the full chain of events isprobably intricately regulated, and we do not rule out the in-volvement of factors other than the NLRP3 inflammasome.Among the impacts of the NLRP3 inflammasome on cell mi-gration, we demonstrated the involvement of IL-1β and IL-18. Itis of note that inflammasomes induce pyroptotic cell death inaddition to maturation of the cytokines. For EAE induction, theinvolvement of cellular contents released by NLRP3 inflamma-some-mediated pyroptotic cell death into the microenvironmentis possible. On the other hand, a recent study showed regulationof actin polymerization by ASC (27), which may also contributeto cell migration. However, our results here showed that thedefective phenotype of cell migration in immunized Nlrp3−/−

mice is very similar to that of Asc−/− mice, i.e., not specific toASC. Therefore, at least in this EAE model, ASC-specific im-pairment of actin polymerization does not seem to play a majorrole in cell migration. In addition, we have shown similar levelsof 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, andCD11c+ cells in Asc−/− mice. The reason for the discrepancy iscurrently unknown.We observed that direct CD4+ T-cell injection into the CNS

induced much milder EAE and demyelination compared withEAE induced by CD4+ T-cell i.v. injection. As shown in thisstudy and an article by another group (28), direct T-cell injectioninto the CNS may not be an aggressive approach to induce EAE,as it may sound. Severe EAE is developed by i.v. CD4+ T-celltransfer but not by direct CD4+ T-cell transfer to the CNS, be-cause i.v. injection allows time and space for transferred T cellsto proliferate in the periphery before they infiltrate into the CNS.In addition, it is widely known that APCs are recruited togetherfrom 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 de-velopment of EAE. In the case of direct T-cell transfer to theCNS, no inflammatory cell expansion in the periphery is expec-ted as well as extra inflammatory cell recruitment from the pe-riphery. In addition, artificial T-cell injection to the CNS istechnically not as efficient as natural T-cell recruitment into the

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Fig. 6. Bypassing the migration process to the CNS enables CD4+ T cells toinduce EAE despite of priming in Asc−/− or Nlrp3−/− mice. (A) Schematicprocedure for the experiments shown in B–G. (B–G) CD4+ T cells wereobtained from spleens of WT, Asc−/−, or Nlrp3−/− mice at 9 d after immuni-zation and transferred (3 × 106 cells per mouse) into sublethally irradiatedWT recipients (B and C) or Rag2−/− recipients (D). CD4+ T cells (1 × 106 cellsper 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 com-bination of both i.c.v. and i.th. injections (G). G also includes negative con-trols 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-celltransfer (n = 5). *P < 0.05.

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CNS. A recent article showed that autoreactive T cells access theCNS via the fifth lumber spinal cord to induce EAE (29). Thisextremely defined route may make artificial cell injections in-efficient because of the requirement of transferred T cells to beprecisely targeted to the defined route for effective elicitation oftheir encephalitogenicity.There are a number of reports that strongly suggest the in-

volvement of inflammasomes in MS development. It is possiblethat activation of the NLRP3 inflammasome induces in-flammatory cell recruitment into the CNS. Our study suggestsa strong connection between the NLRP3 inflammasome andimmune cell migration through induction of chemokines andtheir receptors. As currently applied in clinical interventions ofMS, targeting molecules that enhance immune cell migrationappears to be an effective approach in treating MS accompaniedwith NLRP3 inflammasome activation.

Materials and MethodsAnimals. Male mice of the C57BL/6 background were used in this study. TheAsc−/− and Nlrp3−/− mice were a gift from Genentech and were rederived inour facility. The 2D2 and OT-2 T-cell receptor (TCR) transgenic (Tg) mice werepurchased from The Jackson Laboratory. The mice were kept in a barrierfacility. This study was approved by the Duke University Institutional Animal

Care and Use Committee. EAE induction was performed as previously de-scribed (18).

Adoptive Transfer of CD4+ Th Cells and Th17 Cells. CD4+ T cells, IL-17+, andIFNγ+ 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 mi-crobeads or IL-17- or IFNγ-capture microbeads (Miltenyi Biotec). Isolated Tcells were adoptively transferred by i.v. injection to Rag2−/− recipient mice orsublethally irradiated WT mice (irradiation was performed 24 h before T-celltransfer). Mice were also i.p. injected with pertussis toxin on days 0 and 2. Insome 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 asmean ± SEM.

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

ACKNOWLEDGMENTS. We thank Drs. Tomohiro Arikawa, Yasuhiro Mor-iwaki, Feng Feng, Keitarou Matsumoto, and Masaki Kimura for technicalhelp and Dr. Yuan Zhuang and Yen-yu Lin for MOG tetramer. This work wassupported by grants from the National Multiple Sclerosis Society (to M.L.S.)(RG4536-A-1) and National Institutes of Health (to K.L.W) (AI089756).

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