NLRP3 plays a critical role in the development of experimental autoimmune encephalomyelitis by mediating Th1 and Th17 responses

Abstract

The interplay between innate and adaptive immunity is important in multiple sclerosis (MS). The inflammasome complex, which activates caspase-1 to process pro-IL-1beta and pro-IL-18, is rapidly emerging as a pivotal regulator of innate immunity, with nucleotide-binding domain, leucine-rich repeat containing protein family, pyrin domain containing 3 (NLRP3) (cryopyrin or NALP3) as a prominent player. Although the role of NLRP3 in host response to pathogen associated molecular patterns and danger associated molecular patterns is well documented, its role in autoimmune diseases is less well studied. To investigate the role of NLRP3 protein in MS, we used a mouse model of MS, experimental autoimmune encephalomyelitis (EAE). Nlrp3 expression was elevated in the spinal cords during EAE, and Nlrp3(-/-) mice had a dramatically delayed course and reduced severity of disease. This was accompanied by a significant reduction of the inflammatory infiltrate including macrophages, dendritic cells, CD4, and CD8(+) T cells in the spinal cords of the Nlrp3(-/-) mice, whereas microglial accumulation remained the same. Nlrp3(-/-) mice also displayed improved histology in the spinal cords with reduced destruction of myelin and astrogliosis. Nlrp3(-/-) mice with EAE produced less IL-18, and the disease course was similar to Il18(-/-) mice. Furthermore, Nlrp3(-/-) and Il18(-/-) mice had similarly reduced IFN-gamma and IL-17 production. Thus, NLRP3 plays a critical role in the induction of the EAE, likely through effects on capase-1-dependent cytokines which then influence Th1 and Th17.

FIGURE 1

Impaired CD4⁺ T cell responses in Nlrp3^−/− mice. A, Relative expression of Nlrp3 in different subsets of APCs (pDC; Mϕ; superscript i indicates cells that were purified from spleen and lymph nodes 3 d after immunization). The calculation for relative expression was performed as described in Materials and Methods. B–D, WT and Nlrp3^−/− mice (n = 3) were i.p. immunized with 100 µg OVA emulsified in CFA. Eight days postimmunization, pooled splenocytes were restimulated with OVA protein overnight. IFN-γ⁺, CD4⁺ T cells were enumerated by ELISPOT assay (B), ELISA (C), IFN-γ intracellular staining (D), and IL-17 intracellular staining (E). The numbers beside the outlined areas indicate the percentage of CD4⁺ T cells that were IL-17 or IFN-γ⁺. F, Proliferation of CD4⁺ T cells from WT and Nlrp3^−/− mice using plate-bound anti-CD3 and anti-CD28 Ab stimulation. Mϕ, macrophage; pDC, plasmacytoid DC.

FIGURE 2

Nlrp3^−/− mice have delayed course and reduced severity of EAE. A, mRNA expression of Nlrp3 in the spinal cord during the course of EAE. The label Ova represents negative controls that were immunized with OVA instead of MOG_35–55; “preclin” indicates stage of the EAE before appearance of the symptoms; “peak” indicates the peak of EAE usually at the 3-wk time point. B, Clinical score of WT (▲) and Nlrp3^−/− mice (■) after immunization using MOG_35–55 in CFA. C, Clinical scores of two experiments were evaluated for 7 wk. Clinical scores were evaluated by two observers. Data represented as the mean ± SE; n = 15. Two-way ANOVA followed by Tukey’s test for multiple comparisons were used to analyze statistical differences (p < 0.05 for both B and C).

FIGURE 3

Spinal cords from Nlrp3^−/− mice have more myelin and less astrogliosis compared with WT animals following EAE induction. A, Sections of the lumbar spinal cord from WT (i, iii) and Nlrp3^−/− (ii, iv) mice stained with anti-MBPAb. The calibration bars are 200µm in i and ii (original magnification ×40) and 50 µmin iii and iv (original magnification ×500). B, Western blot of spinal cord homogenates from WT and Nlrp3^−/− mice probed with anti-MBPAb (inset) and the corresponding quantitation of Western blots are shown. C, Sections of the lumbar spinal cord from WT (i, iii) and Nlrp3^−/− (ii, iv) mice stained with anti-MBPAb. The calibration bars are 200 µm in i and ii (original magnification ×40) and 50 µm in iii and iv (original magnification ×500). D, Western blot of spinal cord homogenates from WT and Nlrp3^−/−, mice probed with anti-GFAPAb (inset) and the corresponding quantitation of Western blots are shown. Data are shown as mean ± SE; n = 4; results are representative of two experiments. Statistical significance was measured using Student t test; *p < 0.05.

FIGURE 4

Ag-specific stimulation induced more proinflammatory cytokines in lymph node cell cultures from WT than Nlrp3^−/− animals. Flow cytometry plots and the corresponding quantitation of IFN-γ–positive (A) and IL-17–positive (B) activated T cells in WT and Nlrp3^−/− splenocytes 4 wk after EAE induction stimulated ex vivo with MOG_35–55. Amount of IFN-γ (C) and IL-17(D) in the supernatants of ex vivo-stimulated splenocytes analyzed by ELISA. Induction of IFN-γ (E) and IL-17 (F) mRNA in ex vivo-stimulated splenocytes. G and H, MOG_35–55-specific CD4⁺ T cell response in the context of APCs from WT or Nlrp3^−/− mice: naive CD4⁺ T cells from 2D2 mice were cocultured with MOG_35–55-pulsed APCs from WT or Nlrp3^−/− mice. OVA was used as a negative control Ag. Intracellular staining of IFN-γ (G) and IL-17 (H) in 2D2 T cells are shown. Data are representative of two experiments performed by two independent researchers. Data are shown as the mean ± SD of n = 5; results are representative of at least four experiments. *p < 0.05; Student t test; statistically significant difference relative to controls.

FIGURE 5

Infiltration of macrophages, DCs, and T cells into the spinal cord postimmunization was reduced in Nlrp3^−/− animals. Representative flow cytometry histogram of cells from spinal cords describing the infiltration of Mg and Mϕ: endogenous microglia and infiltration of macrophages (A), infiltration of DCs (B), infiltration of CD8⁺, T cells (C), and infiltration of CD4⁺ T cells (D). Data are shown as the mean ± SD of n = 5. Results are representative of at least four experiments. *p < 0.01; Student t test; statistically significant difference relative to controls. Mϕ, macrophage; Mg, microglia.

FIGURE 6

IL-18 levels are reduced in Nlrp3^−/− mice in the EAE model. A, Levels of serum and spinal cord IL-18 in WT and Nlrp3^−/− mice. B, Same as A, except for levels of IL-1β. C, Progression of EAE in WT, Il18^−/−, and Nlrp3^−/− mice. D, IFN-γ in the spinal cords 3 wk after immunization. E, IL-17 in the spinal cords 3 wk after immunization. F, MCP-1 in the spinal cords 3 wk after immunization. Data are shown as a mean ± SD of n = 5. *p < 0.05; Student t test; statistically significant difference relative to controls.