The AIM2 inflammasome is activated in astrocytes during the late phase of EAE
William E. Barclay, Nupur Aggarwal, M. Elizabeth Deerhake, Makoto Inoue, Toshiaki Nonaka, Kengo Nozaki, Nathan A. Luzum, Edward A. Miao, Mari L. Shinohara
William E. Barclay, Nupur Aggarwal, M. Elizabeth Deerhake, Makoto Inoue, Toshiaki Nonaka, Kengo Nozaki, Nathan A. Luzum, Edward A. Miao, Mari L. Shinohara
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Resource and Technical Advance Neuroscience

The AIM2 inflammasome is activated in astrocytes during the late phase of EAE

Abstract

Inflammasomes are a class of innate immune signaling platforms that activate in response to an array of cellular damage and pathogens. Inflammasomes promote inflammation under many circumstances to enhance immunity against pathogens and inflammatory responses through their effector cytokines, IL-1β and IL-18. Multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE), are autoimmune conditions influenced by inflammasomes. Despite work investigating inflammasomes during EAE, little remains known concerning the role of inflammasomes in the central nervous system (CNS) during the disease. Here, we used multiple genetically modified mouse models to monitor activated inflammasomes in situ based on oligomerization of apoptosis-associated speck-like protein containing a CARD (ASC) in the spinal cord. Using inflammasome reporter mice, we found heightened inflammasome activation in astrocytes after the disease peak. In contrast, microglia and CNS-infiltrated myeloid cells had few activated inflammasomes in the CNS during EAE. Astrocyte inflammasome activation during EAE was dependent on absent in melanoma 2 (AIM2), but low IL-1β release and no significant signs of cell death were found. Thus, the AIM2 inflammasome activation in astrocytes may have a distinct role from traditional inflammasome-mediated inflammation.

Authors

William E. Barclay, Nupur Aggarwal, M. Elizabeth Deerhake, Makoto Inoue, Toshiaki Nonaka, Kengo Nozaki, Nathan A. Luzum, Edward A. Miao, Mari L. Shinohara

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Figure 6

Outcomes of inflammasome activation in astrocytes.

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Outcomes of inflammasome activation in astrocytes. (A and B) RT-qPCR eva...
(A and B) RT-qPCR evaluation of gene expression for Il1b and Il18 (A) and Casp1 and Gsdmd (B) in SC total cells versus astrocytes isolated from naive mice (n = 4) and mice with EAE at 30 dpi (n = 7). Two-way RM ANOVAs were used with Holm-Šidák multiple comparisons test post hoc. Each data point represents a value from 1 mouse. (C) Representative images of Western blotting for inflammasome components in BMDMs versus primary cortical astrocytes. Cells indicated as group 1: unstimulated; group 2: treated with ultrapure LPS alone; group 3: pretreated with ultrapure LPS and stimulated with nigericin to activate the NLRP3 inflammasome; and group 4: pretreated with ultrapure LPS and stimulated with poly(dA:dT)/liposome to activate the AIM2 inflammasome. GSDMD-FL, uncleaved GSDMD; GSDMD-NT, cleaved N-terminal GSDMD. (D–F) Representative images (D and E) and quantification (F) of TUNEL staining of SC sections from naive (n = 3) and 30 dpi EAE ASC-Citrine mice (n = 4). Arrows indicate TUNEL+ cells (E). Two-way RM ANOVA was used (F). Scale bar is 75 μm. (G and H) Representative images (G) and quantification (H) of active caspase-3 (CC3) in SC astrocytes with and without ASC specks/strings. Evaluated from naive (n = 7) and at 30 dpi EAE (n = 6) ASC-Citrine mice. Scale bar is 15 μm. Individual astrocytes were identified using the Imaris software and were quantified by CC3 puncta staining. Each data point represents a value from 1 mouse, combined from multiple experiments. Two-way RM ANOVA was used with Holm-Šidák multiple-comparison test post hoc. (H) **P < 0.01, ***P < 0.001, ****P < 0.0001. Error bars denote mean ± SEM (A, B, F, and H).