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 protein containing a carboxy- three kinds of proteins, 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 chemotaxis-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- chemokine-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 l) 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 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.