Under the low gene expression, we did not observe astrocyte enrichment of and expression, as suggested in the RiboTag data (Supplemental Figure 4A)

Under the low gene expression, we did not observe astrocyte enrichment of and expression, as suggested in the RiboTag data (Supplemental Figure 4A). during EAE. Astrocyte inflammasome activation during EAE was dependent on absent in melanoma 2 (AIM2), but low IL-1 release and no significant indicators of cell death were found. Thus, the AIM2 inflammasome activation in astrocytes may have a distinct role from traditional inflammasome-mediated inflammation. gene expression, suggesting the possibility that AIM2 inflammasome activation in astrocytes serves a different purpose than traditional inflammasome-mediated inflammation. Results Inflammasome activation in the SC at a late stage of EAE. Inflammasome signaling is critical to EAE development in the peripheral lymphoid organs (2C5, 10). Yet, the extent and spatiotemporal distribution of inflammasome activation in the CNS during EAE is largely unknown. Classically, detection of inflammasome activation is performed Mouse monoclonal to Myoglobin by identifying cleaved caspase-1 by Western blotting (WB), which cannot be applied in situ. Therefore, we used a different molecular signature of inflammasome activation the oligomerization of ASC, microscopically observed as the ASC speck. In this study, we used inflammasome activation reporter mice, which express ASC fused to a fluorescent Citrine protein (ASC-Citrine) (19). The ASC-Citrine reporter allows in situ detection of active inflammasomes by visualization of ASC specks (13, 19) (Supplemental Physique 1A; supplemental material available online with this short article; https://doi.org/10.1172/jci.insight.155563DS1), and the presence of the reporter did not alter the disease course of EAE (Physique 1A). The ASC-Citrine reporter was validated for use in tissue by immunostaining against ASC in live spleen slice cultures following NLRP3 inflammasome activation (Supplemental Physique 1A). Open in MRTX1257 a separate window Physique 1 Inflammasome activation in the CNS during late EAE.(A) EAE disease score of WT (= 5) versus ASC-Citrine mice (= 4). Mann-Whitney test of total AUC for disease was used. Each data point represents a imply value among a group. (BCD) Representative images (B and C) and quantification (D) of ASC specks in the iLNs of ASC-Citrine mice during EAE. Each MRTX1257 data point represents an average value from 2 cross sections of both iLNs (25 m thickness) per mouse. = 5 mice. One-way ANOVA, = 0.0006, with Dunnetts multiple comparisons test. Level bar is usually 20 m. (ECG) Representative images of SC from ASC-Citrine mice at indicated time points during EAE. Level bar is usually 300 m. (HCK) Representative images (H and J) and quantification (I and K) of ASC specks (H and I) and ASC strings (J and K) in SC from ASC-Citrine mice during EAE. Level bar is usually 10 m. Each data point represents a value from 1 mouse (= 5). Two coronal cross sections (25 m thickness) of MRTX1257 L5 SC from 1 mouse were quantified manually and averaged. One-way ANOVA with Dunnetts multiple comparisons assessments (D, I, and K). ** 0.01, *** 0.001, **** 0.0001. Error bars denote mean SEM (A, D, I, and K). Before evaluating inflammasome activation in the CNS, we first visualized and quantified ASC specks in the iLNs as the primary site of immune reaction to EAE induction. ASC specks were detected at 3 days postinduction (dpi) of EAE (Physique 1, BCD), which is usually well before the disease onset, and continually detected until 9 dpi (Physique 1D). However, ASC specks were almost undetectable by the point of disease peak (16 dpi) and after (Physique 1D). The cervical lymph nodes have also been noted as a site of main immune reaction in some models of EAE (20, 21), but few ASC specks were detected there throughout disease (Supplemental Physique 1B). The SC of ASC-Citrine mice exhibited a much higher quantity of ASC specks than the iLNs (Physique 1, ECI), with a significant increase in the number of ASC specks in the later phase of EAE at 30 dpi (Physique 1, G and I). Further, in addition to ASC specks, we also observed atypical fiber-like ASC-Citrine signals, which we termed ASC strings, unique to the CNS (Physique 1, J and K). While different in magnitude, both ASC specks and ASC strings appeared with the largest increases at the later phase of disease, around 30 dpi (Physique 1, I and K). Due to the large quantity of ASC specks and strings at 30 dpi, all subsequent analyses in SC were at 30 dpi, unless otherwise stated. This quantification was also performed manually, and all subsequent.