Cutting edge: critical role for PYCARD/ASC in the development of experimental autoimmune encephalomyelitis

Abstract

Multiple sclerosis is an autoimmune disease in which self-reactive T cells attack oligodendrocytes that myelinate axons in the CNS. Experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, is dependent on caspase-1; however, the role of Nod-like receptors upstream of caspase-1 is unknown. Danger- and pathogen-associated molecular patterns activate Nod-like receptor 3, which activates caspase-1 through the adaptor protein, apoptosis-associated speck-like protein containing CARD (ASC). We report that the progression of EAE is dependent on ASC and caspase-1 but not Nod-like receptor 3. ASC(-/-) mice were even more protected from the progression of EAE than were caspase-1(-/-) mice, suggesting that an inflammasome-independent function of ASC contributes to the progression of EAE. We found that CD4(+) T cells deficient in ASC exhibited impaired survival; accordingly, ASC(-/-) mice had fewer myelin oligodendrocyte glycoprotein-specific T cells in the draining lymph nodes and CNS.

Fig. 1. ASC^−/−, but not NLRP3^−/−, mice are protected from EAE

MOG-induced EAE was induced and given clinical scores daily as described in Materials and Methods. The average clinical score of all of the mice from two independent experiments is shown. AC, WT (filled) and A, caspase-1^−/− (open); B, NLRP3^−/− (open); C, ASC^−/− (open) mice were monitored for signs of disease. Data are represented as the average ± SEM. n = 12–18 per group.

Fig. 2. Peripheral and CNS responses of MOG-specific T cells in ASC^−/− mice during EAE induction

WT (black bars) and ASC^−/− (gray bars) mice were killed 10 (A,B,C) or 17 (D,E) days after immunization. A, Splenocytes were harvested and stimulated in vitro with various concentrations of MOG peptide for 48 h. [³H]Thymidine was added for the last 8 h, and its incorporation was measured. The cells were stimulated in triplicate and averaged together. B, Splenocytes were harvested and stimulated with 30 μg/mL MOG peptide for 48 h. The supernatants were harvested and analyzed for cytokine production with a Millipore cytokine bead-plex. C, Cells were harvested from the draining lymph nodes, stimulated with MOG peptide and stained for intracellular cytokines as describe in Materials and Methods. D, Cells were harvested from the spinal cord, stimulated with MOG peptide and stained for intracellular cytokines. The averages are plotted ± SEM. The data are representative of two independent experiments. n = 5. E, Spinal cords were removed 17 days after immunization and stained for CD3⁺ T cells. Spinal cords representative of the group are shown.

Fig. 3. The number of CNS-infiltrating inflammatory cells is decreased in ASC^−/− mice and the peripheral survival of ASC^−/− CD4⁺ T cells is impaired

A, Cells were harvested from the spinal cords of mice 17 days after immunization, stained with surface markers and analyzed by flow cytometry. B, Lethally irradiated WT (CD45.1) mice were injected with bone marrow from WT (CD45.1/2) and ASC^−/− (CD45.2) mice at an equal ratio. The mice were euthanized 6 weeks later and the percentage of WT and ASC^−/− CD4⁺ T cells were measured. C, Mature CD4⁺ T cells were isolated from WT (CD45.1/2) and ASC^−/− (CD45.2) mice and injected into naïve WT (CD45.1) mice at a 1:1 ratio. Mice were bled on designated days and the ratio of injected WT and ASC^−/− CD4⁺ T cells in the blood was determined by flow cytometry. The average is plotted ± SEM. The data are representative of five injected mice.