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 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 upregulate 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.
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neuroinflammation passive experimental autoimmune encephalomyelitis demyelination intrathecal injection intracerebroventricular injection
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xperimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), is mediated by myelinspecific 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. Antigenpresenting 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), 10480–10485 | PNAS | June 26, 2012 | vol. 109 | no. 26
apoptosis-associated speck-like protein containing a carboxyterminal 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. 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
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. 1
To whom correspondence should be addressed. E-mail:
[email protected].
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1201836109/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1201836109
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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. 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.
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). Inoue et al.
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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. IL17+ 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).
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. 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. 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 PNAS | June 26, 2012 | vol. 109 | no. 26 | 10481
IMMUNOLOGY
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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.
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
