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

Makoto 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 of apoptosis-associated speck-like containing a carboxy- three kinds of , NLRP3, ASC, and pro-caspase-1, and plays terminal CARD (ASC), and pro-caspase-1, and found in innate a role in sensing pathogens and danger signals in the innate im- immune cells, such as macrophages and DCs. Active NLRP3 mune system. The NLRP3 inflammasome is thought to be involved inflammasome processes pro–IL-1β and pro–IL-18 to produce in the development of experimental autoimmune encephalomyeli- mature IL-1β and IL-18, respectively. We and another group − − tis (EAE), an animal model of multiple sclerosis (MS). However, the reported that mice lacking genes for Nlrp3 or Asc (Nlrp3 / and − − mechanism by which the NLRP3 inflammasome induces EAE is not Asc / mice) are resistant to the development of EAE (6, 7), clear. In this study, we found that the NLRP3 inflammasome suggesting the association of the NLRP3 inflammasome with played a critical role in inducing T-helper cell migration into the EAE development. In MS plaques and/or cells from MS patients, CNS. To gain migratory ability, CD4+ T cells need to be primed by the expression of caspase-1, IL-1β, and IL-18 is elevated (8–10), NLRP3 inflammasome-sufficient antigen-presenting cells to up- suggesting the involvement of the NLRP3 inflammasome in MS regulate -related proteins, such as osteopontin, CCR2, pathogenicity. However, the mechanism by which the NLRP3 and CXCR6. In the presence of the NLRP3 inflammasome, dendritic inflammasome induces development of EAE and MS is poorly cells and macrophages also induce chemotactic ability and up-reg- understood. In this study, we demonstrate that the NLRP3 ulate chemotaxis-related proteins, such as α4β1 integrin, CCL7, inflammasome in APCs induces EAE development by enhancing CCL8, and CXCL16. On the other hand, reduced Th17 cell popula- -mediated immune cell recruitment in the CNS. In Nlrp3−/− Asc−/− − − − − tion size in immunized and mice is not a determi- contrast, attenuated Th17 response in Nlrp3 / and Asc / mice native factor for their resistance to EAE. As currently applied in is not a determinative factor in their resistance against EAE. clinical interventions of MS, targeting immune cell migration mol- Therefore, inhibiting cell migration may be a good target if ecules may be an effective approach in treating MS accompanied NLRP3 inflammasome activation induces progression of MS. by NLRP3 inflammasome activation. Results fl neuroin ammation | passive experimental autoimmune encephalomyelitis | Asc−/− and Nlrp3−/− Mice Were Resistant to EAE with Decreased demyelination | intrathecal injection | intracerebroventricular injection −/− Immune Cell Infiltration in the CNS. We first observed that Asc − − and Nlrp3 / mice were resistant to EAE (Fig. 1A), as previously − − − − xperimental autoimmune encephalomyelitis (EAE), an ani- reported (6, 7). Because Asc / and Nlrp3 / mice were equally Emal model of multiple sclerosis (MS), is mediated by myelin- resistant to EAE development (Fig. 1A), the NLRP3 inflam- − − specific autoreactive T-helper (Th) cells. Once Th cells are masome appeared to be required for disease progression. Asc / generated, their migration to the CNS is the next important step mice showed little demyelination [Luxol fast blue (LFB) staining, for EAE progression. Th cells infiltrate in the CNS by crossing Fig. 1B] and few infiltrating cells (H&E staining, Fig. 1B) in the the blood–brain barrier and mediate inflammatory responses, spinal cord at the peak of disease (day 17). To enumerate cells resulting in demyelination and neurodegeneration. Antigen- infiltrated in the CNS, day 17 brains and spinal cords were − − − − presenting cells (APCs), such as dendritic cells (DCs) and mac- harvested. Both Asc / and Nlrp3 / mice displayed dramatically rophages, also contribute to the progression of EAE by being reduced numbers of total leukocytes and CD4+ T cells in spinal recruited in the CNS. Together with CNS-resident APCs, cords and brains (Fig. 1 C and D and Fig. S1 A–C). Furthermore, fi recruited APCs restimulate CNS-in ltrated Th cells and even- both Th17 and Th1 cells were almost completely absent in the − − − − tually cause tissue damage together with Th cells in the CNS. CNS of Asc / and Nlrp3 / mice (Fig. 1E and Fig. S1 C). Col- Factors that enhance immune cell migration play a critical role lectively, these data demonstrate that the NLRP3 inflammasome fi in EAE development. For example, mice de cient in CCR2, is required for EAE development, demyelination, and cell re- a major chemokine receptor, show severely compromised cell cruitment in the CNS. migration to spinal cords and are resistant to EAE (1). CCR2 antagonist and a neutralizing antibody for a CCR2 ligand, CCL2, Reduced Th17 Response Does Not Account for EAE Resistance in suppress EAE progression (2, 3). EAE progression is also sup- Immunized Asc−/− and Nlrp3−/− Mice. Inflammasome activation is pressed by treatment with the sphingosine 1-phosphate receptor required for maturation and secretion of IL-1β. We reported 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 Author contributions: M.I. and M.L.S. designed research; M.I. performed research; K.L.W. MS (5). FTY720 is now in clinical trials, and integrin α4 antibody contributed new reagents/analytic tools; M.I., M.D.G., and M.L.S. analyzed data; and M.I. (natalizumab) is currently used to treat MS patients. Targeting and M.L.S. wrote the paper. migration molecules can be quite effective in MS treatments. The authors declare no conflict of interest. The NLRP3 inflammasome senses pathogens and danger *This Direct Submission article had a prearranged editor. signals, such as bacteria, fungi, extracellular ATP, amyloid β, and 1To whom correspondence should be addressed. E-mail: [email protected]. fl uric acid. The NLRP3 in ammasome is a multiprotein complex, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. comprising NLR family, pyrin domain containing 3 (NLRP3), 1073/pnas.1201836109/-/DCSupplemental.

10480–10485 | PNAS | June 26, 2012 | vol. 109 | no. 26 www.pnas.org/cgi/doi/10.1073/pnas.1201836109 Downloaded by guest on October 2, 2021 A B Spinal cord (day 17) A B DLNs (day 9) In vitro (DC+T cells) 3 WT LFB H&E * Asc -/- WT 9.0 2.0 *

2.5 -/- WT ) 5 OT2+IL23, TGFβ,IL6 Nlrp3 5 2 4 WT DC Asc -/- DC Nlrp3 -/- DC 2.9 0.9 1.7 1.5 1.8 3 1 Asc -/- Asc -/- 4.8 0.5 2 EAE score EAE 0.5 1 * 17 Th17 cell (x10 0 - 0.5 0.9 0.9

0.7 IL 0 5 10 15 20 25 30 0 IFNγ Day IL-17 IFNγ

CDSC SC E C D E ) * SC * 3 * Th17 (WT) ) 80 * ) * ) 4 Immunization 4 * 3 2 *

40 3 25 -/- WT 3.1 1.1 ** * Th17 (ASC ) x10 WT 1.5 -/- x10 30 60 3 20 Th17 (Nlrp3 ) Asc-/- s( 8.6 15 40 1 2 Nlrp3-/-

ell 20 Asc -/- 0.3 0.5 (ng/m l) 1.0 T cells ( 1 10 day 9 + 10 20 17

Th1 cell (x10 cell Th1 5 Th17 Th17 cell (x10 cell Th17 1.2 0 IL-

Total c Total 0 * CD4 0 0.5 0 0 i.v. score EAE IL-17 IFNγ 0 0 5 10 15 20 25 30 35 −/− −/− Rag2-/- Fig. 1. Asc and Nlrp3 mice are resistant to EAE. (A) EAE development. Time (day) Representative data from three independent experiments are shown. Dis- −/− ease scores were presented as mean ± SEM for each group (n = 5). (B) LFB Fig. 2. Reduced Th17 response does not account for EAE resistance in Asc − − −/− and H&E staining of spinal cord sections from WT and Asc / mice at 17 d and Nlrp3 mice. (A) Intracellular staining of IL-17 and IFNγ, and numbers after EAE induction. Squares indicate representative regions shown at a high of Th17 cells in DLNs at 9 d after EAE induction (n =4–6). (B) In vitro Th17 cell + magnification on the right. Arrowheads indicate regions of demyelination. generation. OT-2 CD4 T cells were activated by splenic DCs from naïve mice Representative data from three independent experiments are shown. (C and with a Th17-polarizing condition. Flow cytometry plots show IL-17 and IFNγ + D) Numbers of total cells (C) and CD4+ T cells (D) obtained from spinal cords intracellular staining in CD4 T cells. Representative data from three in- − − − − of WT, Asc / , and Nlrp3 / mice at 17 d after EAE induction (n =6–9). dependent experiments are shown. (C) IL-17 concentration in culture Horizontal lines denote mean values. (E) Intracellular staining of IL-17 supernatants from experiments shown in B in triplicate wells. Representative and IFNγ and numbers of Th17 and Th1 cells in spinal cords of WT Asc and data from three independent experiments are shown. (D) Schematic pro- − − + Nlrp3 / mice at 17 d after EAE induction (n =6–8). *P < 0.05. cedure for the experiment shown in E. IL-17 cells were enriched by microbeads from WT, Asc−/−,orNlrp3−/− mice at 9 d after immunization. IL- 17+ cells (1 × 106 cells per mouse) were adaptively transferred into Rag2−/− + abundant levels of serum IL-1β in WT mice with EAE but not in mice. (E) Passive EAE induced by IL-17 cell transfer. Disease scores were − − − − ± immunized Asc / and Nlrp3 / mice (7). Because IL-1β pro- presented as mean SEM for each group (n =5). motes Th17 cell generation (11, 12), we expected that deficiency fl of the NLRP3 in ammasome greatly attenuated Th17 cell pop- Collectively, the quantity (i.e., the reduction) of Th17 population − − − − ulation in our EAE model. However, Th17 cell numbers were does not explain the resistance to EAE in Asc / and Nlrp3 / reduced only about 50% in the draining lymph nodes (DLNs) of − − − − mice, but the data here suggested that the quality of Th17 cells is / / − − − − Asc and Nlrp3 mice (Fig. 2A). Similar levels of partial re- altered in immunized Asc / and Nlrp3 / mice. duction were seen in the proportion of Th17 cells and in con- centrations of IL-17 in culture supernatants after in vitro Immune Cell Accumulation in Peripheral Lymphoid Organs and Blood + −/− −/− stimulation of CD4 T cells by Asc and Nlrp3 DCs (Fig. 2 B in Immunized Mice Deficient in the NLRP3 Inflammasome. Decreased + γδ + −/− −/− and C). We found that numbers of splenic IL-17 T cells were infiltration of CD4 T cells in the CNS of Asc and Nlrp3 −/− −/− reduced about 50% in Asc and Nlrp3 mice as well (Fig. mice may be caused by decreased cellularity in the periphery. To −/− S1D). Numbers of Th1 cell in the DLNs from immunized Asc examine this possibility, we first evaluated cellularity in periph- −/− and Nlrp3 mice were also reduced to about half of those from eral lymphoid organs and in blood on days 0, 9, and 17 after WT mice (Fig. S1E), although proportions of Th1 cells were immunization. Although similar sizes of DLNs and spleens were −/− −/− − − slightly increased in in vitro culture with Asc or Nlrp3 DCs, observed in WT and Asc / mice on days 0 and 9 (Fig. S2), DLNs + − − (Fig. 2B). Thus, numbers of Th17, Th1, and IL-17 γδT cells and spleens were enlarged in Asc / mice at the peak of EAE −/− −/− were reduced in Asc and Nlrp3 mice, but only partially. (day 17) (Fig. 3A, Top). Consistent with the enlargement of Here, we speculated that such partial reduction may not fully spleens and DLNs, numbers of total and myelin oligodendrocyte −/− account for the significant resistance against EAE in Asc and glycoprotein (MOG)-specific CD4+ T cells kept increasing in −/− − − Nlrp3 mice (Fig. 1A). To test whether cell numbers matter, we Asc / mice by day 17 in DLNs and spleen, whereas these focused on the Th17 cell population, because Th17 cells were numbers started reducing in WT mice after day 9 (Fig. 3A and dominant over Th1 cells in DLNs (Fig. 2A and Fig. S1E). We Fig. S3A). Numbers of these cells in blood also showed similar −/− − − obtained Th17 cells from immunized mice (WT, Asc ,or tendency, although the numbers in Asc / mice did not keep −/− Nlrp3 ) by cytokine capture beads and transferred the same increasing after day 9 (Fig. 3A). S1PR1 expressed in T cells + number of Th17 cells to WT hosts. If a reduced number of IL-17 directs their egress from lymph nodes into lymph and recircu- −/− −/− cells is a reason for the EAE resistance in Asc and Nlrp3 lation, but we found no alteration in expression of the S1pr1 gene −/− −/− + mice, recipients transferred with Asc or Nlrp3 IL-17 and S1PR1 protein in total and MOG-specific CD4+ T cells from − − cells should develop EAE (Fig. 2D). However, EAE developed Asc / mice (Fig. S3 B and C), suggesting no defect in T-cell only in WT recipients transferred with Th17 cells but not with egress from lymph nodes. These data demonstrated that NLRP3 −/− −/− Asc and Nlrp3 Th17 cells (Fig. 2E). This result suggests that inflammasome-deficient mice increased cellularity of circulating the reduction of Th17 cells does not account for EAE resistance + − − − − cells, including CD4 T cells, at the disease peak. in immunized Asc / and Nlrp3 / mice. Here, we evaluated the − − − − expression levels of IL-17 in Th17 cells isolated from WT, Asc / , Normal T-Cell Proliferation and Cell Death in Immunized Asc / and − − − − − − and Nlrp3 / mice, but expression levels of IL-17 were similar in Nlrp3−/− Mice. It is not known whether Asc / and Nlrp3 / mice

Th17 cells from these mice (mean fluorescence intensity: WT, have defects in generating and sustaining antigen-specific T cells IMMUNOLOGY − − − − 292.7 ± 0.9; Asc / , 305.3 ± 2.9; Nlrp3 / , 282.6 ± 0.2). during EAE development. Here, we found no significant

Inoue et al. PNAS | June 26, 2012 | vol. 109 | no. 26 | 10481 Downloaded by guest on October 2, 2021 DLNs Spleen * cells because the attenuated gene expression may simply be at- A WT Asc -/- WT Asc -/- C 1 None tributed to the reduction of the Th17 population size in total Day17 rIL-1β + rIL18 CD4 T cells (Fig. 2A). IL-17 capture beads were used to isolate 0.5 IL-17+ cells from spleens. Although Ccr1 and Ccl9 mRNA levels − −

Blood (ng/ml) OPN / 0 turned out to be similar between WT and Asc splenic Th17 250 25 ) 40 * 6 WT * 20 * cells (Fig. S4B), reduced Spp1, Ccr2, and Cxcr6 mRNA expres- 30 Asc -/- 200 20 None 150 15 15 rIL-1β sion was still observed in splenic Th17 cells from immunized 20 * −/− 100 10 rIL18 Asc mice (Fig. 3B). In addition, significant reduction of Spp1 10 WT 10 50 WT 5 -/- Total (x10 Asc -/- Asc and Cxcr6 mRNA expression was also observed in splenic Th1 0 0 0 5 − − CCR2 (MFI) / 0 9 17 0 9 17 0 9 17 ) 0 cells from immunized Asc mice (Fig. 3B). These data suggest 6 10 30 1.5 −/− WT * * * 40 * that Th17 and Th1 cells in immunized Asc mice have a dif- 7.5 Asc -/- * None 20 1.0 30 T (x10 5 rIL-1β ferent gene-expression pattern from that of WT Th17 and Th1 + rIL18 2.5 10 0.5 WT 20 cells, indicating altered quality of Th cells. WT -/- CD4 Asc -/- Asc 0 0 0 10 We then sought to elucidate a molecular mechanism by which 0 9 17 0 9 17 0 9 17 CXCR6 (MFI) fl Day Day Day 0 the NLRP3 in ammasome regulates migration-related genes in Th cells. NLRP3 inflammasome processes maturation of IL-1β B CD4+ T Th17 Th1 D CD4+ (WT) and IL-18. We previously observed the elevated serum IL-1β + -/- WT WT 1.5 WT ) CD4 (Asc ) 1.5 1.5 4 -/- -/- Asc -/- and IL-18 production during EAE progression in WT mice (7). Asc Asc 4 3 1.0 1.0 3 Therefore, we carried out ex vivo experiments to clarify the ex- levels 1.0 2 * β * ** * * * 2 * tent to which IL-1 and IL-18 up-regulate migration-related * * * * + + 0.5 0.5 0.5 1 * 1 * genes in WT CD4 T cells. Naïve WT CD4 T cells were stim-

Relative 0 0 10 100 ulated with CD3/CD28 antibodies with or without recombinant 0 0 0 1 2 20 200

Migrated cell (x10 rCCL2 rCXCL16 β β (ng/ml) (ng/ml) (r)IL-1 or rIL-18. rIL-1 greatly enhanced mRNA expression of Spp1 and Cxcr6 and protein expression of osteopontin (OPN; Fig. 3. Attenuated expression of genes encoding migration-related pro- Spp1 product) and CXCR6 (Fig. 3C and Fig. S4 C–E). rIL-18 teins impairs CD4+ T-cell migration in immunized Asc−/− mice. (A Top)DLNs also significantly enhanced mRNA expression of Ccr2 and and spleens from WT and Asc−/− mice at 17 d after EAE induction. (Middle Cxcr6 and protein expression of CCR2 and CXCR6 (Fig. 3C and Bottom) Numbers of total cells (Middle) and CD4+ T cells (Bottom)inthe and Fig. S4 C–E). −/− DLNs, spleens, and peripheral blood in WT and Asc mice on the indicated Because expression of Ccr2 and Cxcr6 is decreased in CD4+ days after EAE induction (n =6–11). (B) Gene expression determined by qPCR −/− + + T cells from immunized Asc mice (Fig. 3B and Table S1), 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 CD4 T-cell chemotaxis toward CCL2 (CCR2 ligand) and rIL-18 (100 ng/mL) in tissue culture. Protein levels at 24 h after stimulation CXCL16 (CXCR6 ligand), respectively, was evaluated by were determined by ELISA (secreted OPN) and FACS (CCR2 and CXCR6) (n = a Transwell assay. Significantly reduced chemotaxis toward both + 4). Representative FACS data are presented in Fig. S4E.(D) CD4+ T-cell che- rCCL2 and rCXCL16 were observed in CD4 T cells from im- − − motaxis toward rCCL2 or rCXCL16 of indicated concentrations evaluated by munized Asc / mice (Fig. 3D), suggesting that attenuated gene a Transwell assay of triplicate wells. (B and D) Cells were obtained from expression of Ccr2 and Cxcr6 in CD4+ T cells from immunized −/− − − spleens of WT or Asc mice at 9 d postimmunization. Representative data Asc / mice abated T-cell chemotaxis. These results confirm the < from two independent experiments are shown. *P 0.05. critical involvement of ASC for Th cell migration by enhancing migration-related gene expression in the cells. −/− −/− difference between WT and Asc (or Nlrp3 ) mice in pro- NLRP3 Inflammasome Increases the Expression of Genes Encoding fi + portions and absolute numbers of MOG-speci c CD4 T cells Matching Chemokine/Receptor Pairs Between CD4+ T Cells and APCs. on day 9 (Fig. S3A) and in in vivo T-cell proliferation (Fig. S3D). + −/− −/− We examined gene expression in CD4 T cells, but the impaired Asc and Nlrp3 DCs also similarly proliferated MOG- and cellular ability to migrate into the CNS was not limited to CD4+ fi + ovalbumin (OVA)-speci c CD4 T cells ex vivo (Fig. S3E). T cells (Fig. 1C and Fig. S1A). Therefore, we asked whether fi + There was no signi cant difference in CD4 T-cell necrosis and macrophages and DCs attenuated expression of the genes that apoptosis as well (Fig. S3F). These results demonstrate that the encodes matching chemokine/receptor counterparts of OPN, proliferation and cell death of T cells is normal in the peripheral CCR2, and CXCR6. The α4β1 integrin is a receptor for OPN; −/− −/− lymphoid organs of Asc and Nlrp3 mice and suggest a de- CCL2, CCL7, and CCL8 are ligands of CCR2; and CXCL16 is fect in cell migration in these mice. a ligand of CXCR6. Significant reductions in mRNA levels of Itga4, Itgb1, Ccl7, Ccl8, and Cxcl16, but not Ccl2, were identified, fl − − NLRP3 In ammasome Increases Migration-Related Gene Expression particularly in macrophages from Asc / mice at 9 d after im- and Chemotaxis of Th Cells. To determine whether T cells activated − − − − munization (Fig. 4 A and B and Fig. S4F). Furthermore, in tissue in Asc / and Nlrp3 / mice display an alteration in gene ex- β + culture, we found that rIL-1 enhanced expression of Itga4 in pression, we performed a microarray analysis using CD4 T cells DCs; of Ccl2, Ccl7 and Cxcl16 in macrophages and DCs; and of −/− −/− from DLNs and spleens of Asc , Nlrp3 , and WT mice at 9 d Ccl8 in macrophages (Fig. 4 C and D and Fig. S4G). rIL-18 after immunization. Day 9 is the time of EAE onset in WT mice; enhanced Itga4, Ccl2, Ccl7, and Ccl8 in DCs and macrophages therefore, we considered that Th cell migration into the CNS is and Cxcl16 in macrophages (Fig. 4 C and D and Fig. S4G). Al- ongoing on day 9. A majority of genes with great expression though the result left a possibility that some factors other than −/− −/− reduction in Asc and Nlrp3 mice turned out to encode rIL-1β or rIL-18 also play a role in the induction of gene ex- , their receptors, and integrins. Migration-related pression, IL-1β and IL-18 have a significant impact on up-regu- genes that showed <50% expression in either DLNs or spleens of − − − − lating expression of a majority of genes that were examined. Asc / and Nlrp3 / mice included Spp1, Ccr2, Ccl9, Ckap2, Ccl6, To evaluate a functional consequence of attenuated gene ex- − − Ccr1, Ccl8, Vcam1, Cxcr6, Ccr6, and Ccr8 (Table S1). Expression pression of Itga4 and Itgb1 in Asc / DCs, we examined DC levels of Spp1, Ccr2, Ccr1, Ccl9, and Cxcr6 genes were confirmed chemotaxis toward OPN by using DCs harvested from spleens in to be significantly lower in splenic CD4+ T cells in immunized mice at day 9 postimmunization. DCs from immunized WT mice − − Asc / mice compared with those in immunized WT mice (Fig. successfully migrated toward rOPN in an integrin α4-dependent 3B and Fig. S4A). We then examined gene expression in Th17 manner (Fig. 4E and Fig. S4H). On the other hand, DCs from

10482 | www.pnas.org/cgi/doi/10.1073/pnas.1201836109 Inoue et al. Downloaded by guest on October 2, 2021 A B E WT DC A DC B WT C WT CD4+ Macrophage DC + WT WT DC w/ IgG CD4 T Asc-/- -/- + WT ) 2.5 Asc CD4

-/- 4 1.5 -/- 1.5 Asc WT DC w/ α4 Ab Asc Asc-/- DC 1.0 2.0 10 CD4+ T 1.0 1.0 1.5 * i.v. * 5 0.5 1.0

-/- score EAE 0.5 * * * 0.5 * Rag2 0.5 * *

* Relative levels Relative levels * * 0 0 0 0 0 0 10 20 30 Migrated cell (10 10 100 1000 Immunization Time (day) rOPN (ng/ml)

D E CFSE-CD4+ T cells C Macrophage Ccl8 Cxcl16 SC Brain 1.5 Itga4 10 Ccl7 15 * 2 None 2D2Tg * * * * ) * * * * rIL-1β 3 15 12 * 1.0 10 rIL-18 -/- 5 1 WT, Asc 10 8 0.5 5 CD4+ T Nlrp3 -/- * * * 5 4

0 0 0 0 CFSE- i.v. Cells (x10 0 0 Relative levels 624 hr 624 hr 6 24 hr 624 hr CD4+ T - 2 day D Immunization DC Itga4 Ccl7 Ccl8 Cxcl16 Host mice Host mice 3 ** 10 2.5 2 None * * rIL-1β 2.0 * Fig. 5. Presence of the NLRP3 inflammasome in APCs is sufficient to elicit 2 * 1.5 rIL-18 1 5 1.0 T-cell migration. (A) Expression levels of genes encoding NLRP3 inflamma- 1 * 0.5 some components in DCs and CD4+ T cells were determined by qPCR. DCs and 0 0 0 0 CD4+ T cells were obtained from spleen of WT mice at 9 d after immunization Relative levels 624 hr 624 hr 624 hr 624 hr (n = 4). (B) Schematic procedure for the experiment shown in C. CD4+ T cells − − − − Fig. 4. DCs and macrophages from immunized Asc / mice show attenu- were isolated from spleens and lymph nodes of WT and Asc / naïve donor − − ated expression of genes encoding chemokines or chemokine receptors. (A– mice and transferred (1 × 106 cells) into Rag2 / recipients followed by MOG/ D) Gene expression in macrophage (A and C) and DCs (B and D). Bone complete Freund’s adjuvant (CFA) immunization. (C) EAE scores were pre- marrow-derived macrophages (C) and DCs (D) were treated with or without sented as mean ± SEM for each group (n = 5). (D) Schematic procedure for rIL-1β (10 ng/mL) or rIL-18 (100 ng/mL) and harvested at the indicated time the experiment shown in E. Naïve CD4+ 2D2 T cells were labeled with CFSE points. mRNA levels were determined by qPCR (n = 4). *P < 0.05. (E)DC and transferred into WT, Asc−/−,orNlrp3−/− mice that had been immunized chemotaxis toward rOPN of indicated concentrations (n = 4). Integrin α4 at 2 d before the transfer. CD4+ T cells infiltrated into spinal cords and brains antibody or control IgG was incubated with DCs for 1 h, and then DCs were were enumerated at 4 d after the transfer. (E) Cell numbers of infiltrated plated in upper chamber of a Transwell. (A, B, and E) Cells were obtained CFSE-labeled CD4+ T cells into the spinal cord and brain (n = 5). *P < 0.05. − − from spleens of WT or Asc / mice at 9 d after EAE induction. *P < 0.05 compared with WT DC data. − − − − and adoptively transferred into WT, Asc / ,orNlrp3 / hosts that D −/− had been preimmunized with MOG antigen (Fig. 5 ). Migration immunized Asc mice failed to migrate toward OPN (Fig. 4E of CFSE-labeled 2D2 T cells was evaluated at 4 d after the β + and Fig. S4H). In summary, IL-1 and IL-18 up-regulate critical transfer. Although numbers of CFSE-labeled CD4 T cells were mediators of migration both in APCs and Th cells. The media- similar in DLNs and spleens in all of the groups (Fig. S5), only tors included matching ligand/receptor combinations between WT hosts successfully recruited CFSE-labeled CD4+ T cells into T cells and APCs, such as CCR2 (T cells)/CCL7, CCL8, and the spinal cord and the brain (Fig. 5E). This result strongly sug- CCL2 (APCs); CXCR6 (T cells)/CXCL16 (APCs); and OPN gests that the presence of the NLRP3 inflammasome in T-cell– (T cells)/α4β1 integrin (APCs). priming APCs is essential for T-cell migration into the CNS.

− − − − NLRP3 Inflammasome-Dependent Migration Defects Are Not T-Cell– I.v. Transfer of Th Cells Primed in Asc / or Nlrp3 / Mice Does Not −/− Intrinsic. The defects in T-cell migration seen in Asc and Induce EAE, but Direct Transfer to the CNS Does. The findings above −/− − − − − Nlrp3 mice may be attributable to a loss of ASC or NLRP3 suggest that Th cells primed in Asc / or Nlrp3 / mice are not within the T cells themselves or to a loss of inflammasome ac- encephalitogenic because of their inability to migrate into the CNS. tivity within the APCs that stimulate T cells. WT CD4+ T cells To test T-cell migration, CD4+ T cells obtained from immunized − − − − expressed markedly less Nlrp3 and Casp1 mRNA than DCs do WT, Asc / ,orNlrp3 / mice at the time of EAE onset (day 9) − − (Fig. 5A). Because particularly low expression levels of the Nlrp3 were i.v. transferred to irradiated WT or Rag2 / recipients (Fig. gene suggested little NLRP3 inflammasome activity in CD4+ 6A). CD4+ T cells obtained from immunized WT mice induced T cells, we examined the possible impact of ASC expression on passive EAE and infiltrated into the CNS in irradiated WT CD4+ T-cell encephalitogenicity. Naïve CD4+ T cells from WT recipients (Fig. 6 B and C), but CD4+ T cells from immunized − − − − − − − − or Asc / mice were adoptively transferred into Rag2 / recipi- Asc / or Nlrp3 / mice failed to do so. Resistance to EAE by − − ents (Asc+/+), then the recipients were immunized to induce passive transfer of CD4+ T cells from immunized Asc / and − − − − − − − − EAE (Fig. 5B). Rag2 / recipients transferred with naïve Asc / Nlrp3 / mice was also observed in Rag2 / recipient mice CD4+ T cells developed EAE to the same extent as WT CD4+ (Fig. 6D). − − T-cell–transferred Rag2 / recipients did (Fig. 5C). These results Next, we directly transferred CD4+ T cells into the brain or ruled out the involvement of the T-cell–intrinsic ASC and pos- spinal cord by i.c.v. or intrathecal (i.th.) injection, respectively, to sible assembly of the NLRP3 inflammasome in CD4+ T cells bypass the cell migration process. CD4+ T cells from immunized − − − − during EAE development. WT, Asc / ,orNlrp3 / mice developed similar levels of EAE (Fig. 6 E and F). When CD4+ T cells were transferred into both CD4+ T Cells Need to Be Primed by APCs That Express NLRP3 In- the brain and spinal cord, EAE was more severe than with i.c.v. − − flammasome for Migration to the CNS. On the basis of the above or i.th. injection alone, and CD4+ T cells from immunized Asc / − − results, we hypothesized that Th cells need to be primed by APCs or Nlrp3 / mice again induced similar levels of EAE (Fig. 6G), expressing the NLRP3 inflammasome to migrate to the CNS. To as well as demyelination in spinal cord (Fig. S6), as WT CD4+ test this hypothesis, we examined in vivo activation of WT CD4+ T cells did. Congruent with EAE scores, demyelination by i.c.v. −/− −/− + + T cells in Asc and Nlrp3 hosts. Naïve 2D2 CD4 T cells and i.th. transfer of CD4 T cells was milder than that in im- IMMUNOLOGY − − were labeled with carboxyfluorescein succinimidyl ester (CFSE) munized WT mice or Rag2 / with i.v. transfer of CD4+ T cells

Inoue et al. PNAS | June 26, 2012 | vol. 109 | no. 26 | 10483 Downloaded by guest on October 2, 2021 Immunization integrins, including the α4β1 integrin, OPN plays a role in A Day 9 i.v. (B-D) attracting immune cells (15). In addition to high expression of + −/− CD4 T i.c.v (E) OPN in MS lesions (16), Spp1 mice develop milder EAE than i.th. (F) Irradiated B6 (B,C) – α β -/- WT mice did (16 19). The blockade of 4 1 integrin was also WT, Asc i.c.v, i.th. (G) Rag2-/- (D), B6 (E-G) Nlrp3 -/- shown to reduce relapse rates in relapsing–remitting MS patients and to delay progression of the disease (20). CCL2 is one of the B C D − − − − CD4+ (WT) CD4+ (WT) / / + -/- SC + -/- CCR2 ligands. Ccr2 and Ccl2 mice both show reduced CD4 (ASC ) ) CD4 (ASC ) CD4+ (Nlrp3 -/-) 3 20 * 2.5 CD4+ (Nlrp3 -/-) mononuclear cell infiltrate in the CNS with decreased suscepti- 3 * 15 2.0 bility to EAE (1, 21). Previous studies showed that CCR2 ex- 2 + 10 1.5 pression in circulating CD4 T cells is significantly elevated

T cell (x 10 1.0 * + 5 during MS relapse (22, 23). CXCR6 is required for neuro- EAE score EAE EAE score EAE 1 * 0.5 inflammation by immune cell infiltration in cortical injury sites CD4 0 − − 0 0 / 0 15 20 30 40 0 10 20 30 (24). Although Cxcr6 mice develop EAE to a similar extent as Time (day) Time (day) WT mice, antibodies against CXCL16 are known to reduce EAE E F G severity (24, 25). Th1 cell trafficking is reported to be in- 1.0 CD4+ (WT) 1.0 CD4+ (WT) 1.5 CD4+ (WT) dependent of the α4 integrin (26), but for the optimal expression CD4+ (ASC -/-) CD4+ (ASC -/-) CD4+ (ASC -/-) CD4+ (Nlrp3 -/-) CD4+ (Nlrp3 -/-) CD4+ (Nlrp3 -/-) of chemotactic molecules, such as Spp1 and Cxcr6, the NLRP3 1.0 inflammasome is still needed. Indeed, Th1 cells were not 0.5 − − − − 0.5 detected in the CNS of immunized Asc / and Nlrp3 / mice.

EAE score EAE 0.5 Therefore, despite the different migration machinery of Th1 cells from that of Th17 cells, CNS infiltration of both Th subsets 0 0 0 − − − − 0 10 20 30 0 10 20 30 0 10 20 30 / / Time (day) Time (day) Time (day) is greatly compromised in immunized Asc and Nlrp3 mice. In summary, the NLRP3 inflammasome up-regulates expression Fig. 6. Bypassing the migration process to the CNS enables CD4+ T cells to of migration-enhancing molecules (summarized in Fig. S7), − − − − induce EAE despite of priming in Asc / or Nlrp3 / mice. (A) Schematic which are involved in development of EAE and probably in MS procedure for the experiments shown in B–G.(B–G) CD4+ T cells were −/− −/− as well. obtained from spleens of WT, Asc ,orNlrp3 mice at 9 d after immuni- Our study further showed that T cells need to be primed in zation and transferred (3 × 106 cells per mouse) into sublethally irradiated −/− + 6 NLRP3 inflammasome-sufficient mice to migrate into the CNS WT recipients (B and C)orRag2 recipients (D). CD4 T cells (1 × 10 cells per mouse) were also transferred directly into the brains (E) or spinal cords and induce EAE (Fig. S7), although the full chain of events is (F) of WT recipients by i.c.v. or i.th. injection, respectively, or by the com- probably intricately regulated, and we do not rule out the in- bination of both i.c.v. and i.th. injections (G). G also includes negative con- volvement of factors other than the NLRP3 inflammasome. trols with splenic CD4+ T cells from naïve WT (△), Asc−/− (▲)orNlrp3−/− (◆) Among the impacts of the NLRP3 inflammasome on cell mi- mice transferred (1 × 106 cells per mouse) to recipients (no EAE developed). gration, we demonstrated the involvement of IL-1β and IL-18. It (B and D–G) EAE scores were presented as mean ± SEM for each group (n = is of note that inflammasomes induce pyroptotic cell death in + + 5). (C) Numbers of CD4 T cells in spinal cords on day 44 after CD4 T-cell addition to maturation of the cytokines. For EAE induction, the < transfer (n = 5). *P 0.05. involvement of cellular contents released by NLRP3 inflamma- some-mediated pyroptotic cell death into the microenvironment is possible. On the other hand, a recent study showed regulation (Fig. S6). These data suggest that the cell migration is indeed of actin polymerization by ASC (27), which may also contribute the determinative factor for NLRP3 inflammasome-mediated to cell migration. However, our results here showed that the EAE development. − − defective phenotype of cell migration in immunized Nlrp3 / −/− fi Discussion mice is very similar to that of Asc mice, i.e., not speci cto − − ASC. Therefore, at least in this EAE model, ASC-specific im- This study and previous ones (6, 7, 13) showed that Nlrp3 / mice pairment of actin polymerization does not seem to play a major are resistant to the development of EAE, suggesting the asso- role in cell migration. In addition, we have shown similar levels ciation of the NLRP3 inflammasome with EAE development. −/− of cellularity in splenocytes and lymph nodes among naïve WT, We also showed that Asc mice were resistant to EAE as −/− −/− −/− fl Asc , and Nlrp3 mice. In contrast, Ippagunta et al. (27) Nlrp3 mice. Because it is not clear how the NLRP3 in am- showed greatly reduced cellularity in T cells, B cells, and − − masome enhances EAE, we sought to elucidate the mechanism CD11c+ cells in Asc / mice. The reason for the discrepancy is in this study. Currently, the attenuated Th17 cell responses are currently unknown. suggested to be a major underlying mechanism for the resistance We observed that direct CD4+ T-cell injection into the CNS of knockout mice to EAE (13, 14). Indeed, a number of studies induced much milder EAE and demyelination compared with demonstrated the critical role of Th17 responses in EAE de- EAE induced by CD4+ T-cell i.v. injection. As shown in this velopment and the promotion of Th17 cell generation by IL-1β. − − study and an article by another group (28), direct T-cell injection Based on the reduced Th17 population in immunized Asc / and −/− into the CNS may not be an aggressive approach to induce EAE, Nlrp3 mice, it is quite reasonable to consider that Th17 as it may sound. Severe EAE is developed by i.v. CD4+ T-cell mediates the impact of the NLRP3 inflammasome on EAE de- transfer but not by direct CD4+ T-cell transfer to the CNS, be- velopment. However, we found that the reduction of the Th17 cause i.v. injection allows time and space for transferred T cells −/− population does not account for the resistance to EAE in Asc to proliferate in the periphery before they infiltrate into the CNS. −/− and Nlrp3 mice. Instead, the NLRP3 inflammasome is re- In addition, it is widely known that APCs are recruited together quired for Th17 cells to enhance their migration ability to the from the periphery to the CNS in passive EAE induced by i.v. CNS. Not only Th17 cells but APCs and Th1 cells were also T-cell transfer. APCs infiltrated in the CNS restimulate CNS- found to enhance chemotaxis by the NLRP3 inflammasome. infiltrated autoreactive T cells, further contributing to the de- Our data from microarray and quantitative PCR (qPCR) velopment of EAE. In the case of direct T-cell transfer to the analyses showed that Th cells (and the Th17 cell population CNS, no inflammatory cell expansion in the periphery is expec- − − − − alone) from immunized Asc / and Nlrp3 / mice showed less ted as well as extra inflammatory cell recruitment from the pe- expression of several migration-related molecules, such as Spp1, riphery. In addition, artificial T-cell injection to the CNS is Ccr2, and Cxcr6. Spp1 encodes OPN. As a ligand of various technically not as efficient as natural T-cell recruitment into the

10484 | www.pnas.org/cgi/doi/10.1073/pnas.1201836109 Inoue et al. Downloaded by guest on October 2, 2021 CNS. A recent article showed that autoreactive T cells access the Care and Use Committee. EAE induction was performed as previously de- CNS via the fifth lumber spinal cord to induce EAE (29). This scribed (18). extremely defined route may make artificial cell injections in- + + + efficient because of the requirement of transferred T cells to be Adoptive Transfer of CD4 Th Cells and Th17 Cells. CD4 T cells, IL-17 ,and γ+ −/− −/− precisely targeted to the defined route for effective elicitation of IFN cells were isolated from spleens and DLNs of WT, Asc ,orNlrp3 mice at 9 d after EAE induction by positive selection by using CD4 mi- their encephalitogenicity. crobeads or IL-17- or IFNγ-capture microbeads (Miltenyi Biotec). Isolated T − − There are a number of reports that strongly suggest the in- cells were adoptively transferred by i.v. injection to Rag2 / recipient mice or volvement of inflammasomes in MS development. It is possible sublethally irradiated WT mice (irradiation was performed 24 h before T-cell that activation of the NLRP3 inflammasome induces in- transfer). Mice were also i.p. injected with pertussis toxin on days 0 and 2. In flammatory cell recruitment into the CNS. Our study suggests some experiments, isolated CD4+ Th cells were adoptively transferred by i.th. a strong connection between the NLRP3 inflammasome and and/or i.c.v. injection to WT mice with i.p. injection of pertussis toxin on day immune cell migration through induction of chemokines and −4, −2, 0, and 2 (where day 0 is T-cell transfer). their receptors. As currently applied in clinical interventions of MS, targeting molecules that enhance immune cell migration Statistical Analysis. Statistical analysis was performed with Student’s t tests. fi < appears to be an effective approach in treating MS accompanied The criterion of signi cance was set as P 0.05. All results are expressed as mean ± SEM. with NLRP3 inflammasome activation. All other methods and further details are provided in SI Materials and Materials and Methods Methods. Primer sequences are shown in Table S2. Animals. Male mice of the C57BL/6 background were used in this study. The −/− −/− ACKNOWLEDGMENTS. We thank Drs. Tomohiro Arikawa, Yasuhiro Mor- Asc and Nlrp3 mice were a gift from Genentech and were rederived in iwaki, Feng Feng, Keitarou Matsumoto, and Masaki Kimura for technical our facility. The 2D2 and OT-2 T-cell receptor (TCR) transgenic (Tg) mice were help and Dr. Yuan Zhuang and Yen-yu Lin for MOG tetramer. This work was purchased from The Jackson Laboratory. The mice were kept in a barrier supported by grants from the National Multiple Sclerosis Society (to M.L.S.) facility. This study was approved by the Duke University Institutional Animal (RG4536-A-1) and National Institutes of Health (to K.L.W) (AI089756).

1. Fife BT, Huffnagle GB, Kuziel WA, Karpus WJ (2000) CC chemokine receptor 2 is 16. Chabas D, et al. (2001) The influence of the proinflammatory cytokine, osteopontin, critical for induction of experimental autoimmune encephalomyelitis. J Exp Med 192: on autoimmune demyelinating disease. Science 294:1731–1735. 899–905. 17. Jansson M, Panoutsakopoulou V, Baker J, Klein L, Cantor H (2002) Cutting edge: 2. Brodmerkel CM, et al. (2005) Discovery and pharmacological characterization of Attenuated experimental autoimmune encephalomyelitis in Eta-1/osteopontin-de- – a novel rodent-active CCR2 antagonist, INCB3344. J Immunol 175:5370 5378. ficient mice. J Immunol 168:2096–2099. 3. dos Santos AC, et al. (2005) CCL2 and CCL5 mediate leukocyte adhesion in experi- 18. Shinohara ML, Kim JH, Garcia VA, Cantor H (2008) Engagement of the type I in- — mental autoimmune encephalomyelitis An intravital microscopy study. J Neuro- terferon receptor on dendritic cells inhibits T helper 17 cell development: Role of immunol 162:122–129. intracellular osteopontin. Immunity 29:68–78. 4. Kataoka H, et al. (2005) FTY720, sphingosine 1-phosphate receptor modulator, 19. Hur EM, et al. (2007) Osteopontin-induced relapse and progression of autoimmune ameliorates experimental autoimmune encephalomyelitis by inhibition of in- brain disease through enhanced survival of activated T cells. Nat Immunol 8:74–83. filtration. Cell Mol Immunol 2:439–448. 20. Steinman L (2005) Blocking adhesion molecules as therapy for multiple sclerosis: 5. Yednock TA, et al. (1992) Prevention of experimental autoimmune encephalomyelitis Natalizumab. Nat Rev Drug Discov 4:510–518. by antibodies against α4β1 integrin. Nature 356:63–66. 21. Huang DR, Wang J, Kivisakk P, Rollins BJ, Ransohoff RM (2001) Absence of monocyte 6. Gris D, et al. (2010) NLRP3 plays a critical role in the development of experimental autoimmune encephalomyelitis by mediating Th1 and Th17 responses. J Immunol chemoattractant protein 1 in mice leads to decreased local macrophage recruitment fi 185:974–981. and antigen-speci c T helper cell type 1 in experimental autoim- 7. Inoue M, et al. (2012) Interferon-β therapy against EAE is effective only when de- mune encephalomyelitis. J Exp Med 193:713–726. velopment of the disease depends on the NLRP3 inflammasome. Sci Signal 5:ra38. 22. Sørensen TL, Sellebjerg F (2001) Distinct chemokine receptor and cytokine expression 8. Ming X, et al. (2002) Caspase-1 expression in multiple sclerosis plaques and cultured profile in secondary progressive MS. Neurology 57:1371–1376. glial cells. J Neurol Sci 197:9–18. 23. Misu T, et al. (2001) Chemokine receptor expression on T cells in blood and cere- 9. Huang WX, Huang P, Hillert J (2004) Increased expression of caspase-1 and in- brospinal fluid at relapse and remission of multiple sclerosis: Imbalance of Th1/Th2- terleukin-18 in peripheral blood mononuclear cells in patients with multiple sclerosis. associated chemokine signaling. J Neuroimmunol 114:207–212. Mult Scler 10:482–487. 24. Kim JV, et al. (2010) Two-photon laser scanning microscopy imaging of intact spinal 10. Gutierrez EG, Banks WA, Kastin AJ (1994) Blood-borne interleukin-1 receptor an- cord and cerebral cortex reveals requirement for CXCR6 and neuroinflammation in – tagonist crosses the blood-brain barrier. J Neuroimmunol 55:153 160. immune cell infiltration of cortical injury sites. J Immunol Methods 352:89–100. β 11. Acosta-Rodriguez EV, Napolitani G, Lanzavecchia A, Sallusto F (2007) Interleukins 1 25. Fukumoto N, et al. (2004) Critical roles of CXC chemokine ligand 16/scavenger re- and 6 but not transforming growth factor-β are essential for the differentiation of ceptor that binds phosphatidylserine and oxidized lipoprotein in the pathogenesis of interleukin 17-producing human T helper cells. Nat Immunol 8:942–949. both acute and adoptive transfer experimental autoimmune encephalomyelitis. 12. Andrade-Silva L, Ferreira-Paim K, Silva-Vergara ML, Pedrosa AL (2010) Molecular J Immunol 173:1620–1627. characterization and evaluation of virulence factors of Cryptococcus laurentii and 26. Rothhammer V, et al. (2011) Th17 traffic to the central nervous system Cryptococcus neoformans strains isolated from external hospital areas. Fungal Biol α – 114:438–445. independently of 4 integrin expression during EAE. J Exp Med 208:2465 2476. fl 13. Jha S, et al. (2010) The inflammasome sensor, NLRP3, regulates CNS inflammation and 27. Ippagunta SK, et al. (2011) The in ammasome adaptor ASC regulates the function of demyelination via caspase-1 and interleukin-18. J Neurosci 30:15811–15820. adaptive immune cells by controlling Dock2-mediated Rac activation and actin po- 14. Lalor SJ, et al. (2011) Caspase-1–processed cytokines IL-1β and IL-18 promote IL-17 lymerization. Nat Immunol 12:1010–1016. production by γδ and CD4 T cells that mediate autoimmunity. J Immunol 186: 28. McGeachy MJ, et al. (2007) TGF-β and IL-6 drive the production of IL-17 and IL-10 by 5738–5748. T cells and restrain TH-17 cell-mediated pathology. Nat Immunol 8:1390–1397. 15. Uede T (2011) Osteopontin, intrinsic tissue regulator of intractable inflammatory 29. Arima Y, et al. (2012) Regional neural activation defines a gateway for autoreactive diseases. Pathol Int 61:265–280. T cells to cross the blood-brain barrier. Cell 148:447–457. IMMUNOLOGY

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