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. 2018 May 23;9:2036. doi: 10.1038/s41467-018-04376-5

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 5 — Microglia A20 deficiency aggravates autoimmune CNS inflammation due to Nlrp3 inflammasome hyperactivation. a, b EAE was induced by active immunization of control A20^FL (n = 6) and A20^Cx3Cr1-KO mice (n = 10) with MOG peptide, and clinical disease development (a) and body weight (b) was followed over time. Each data point represents the mean ± SEM as estimated by the REML analysis. Changes in clinical score and relative body weight differ significantly (***p < 0.001; F-test) between genotypes across the time span. Data are representative of three independent experiments. c Representative images of spinal cord of control (A20^FL) and A20^Cx3Cr1-KO mice 13 days post immunization using LFB, APP, CD3, and MAC-3 antibodies. Scale bars represent 200 µm (overview) and 20 µm (zoom). Representative images from at least four mice per group are displayed. d Assessment of immune cell (CD3, CD4, and CD8 T cell, Treg) infiltration in the CNS of control (A20^FL) and A20^Cx3Cr1-KO mice by flow cytometry just before disease onset. Each symbol represents one mouse. Data are expressed as mean ± SEM and significant differences are determined by a Mann–Whitney U-statistical test (***p < 0.001, ****p < 0.0001). e Expression of inflammasome-associated factors in the spinal cord of TAM-injected control (A20^FL) and A20^Cx3Cr1-KO mice 12 days post immunization. Each symbol represents one mouse. Data are expressed as the ratio of the mRNA expression normalized to endogenous housekeeping genes and expressed as mean ± SEM. Significant differences are determined by a Mann–Whitney U-statistical test (****p < 0.0001). f, g Active immunization of control (A20^FL, n = 11), A20^Cx3Cr1-KO (n = 10), Nlrp3^KO (n = 18), and A20^Cx3Cr1-KO-Nlrp3^KO (n = 8) mice (f) and of control (A20^FL, n = 28), A20^Cx3Cr1-KO (n = 15) and A20/caspase-1^Cx3Cr1-KO (n = 13) mice (g), and clinical disease development over time. Each data point represents the mean ± SEM as estimated by the REML analysis. Changes in clinical score and relative body weight differ significantly between genotypes across the time span (*p < 0.05, **p < 0.01, ***p < 0.001; F-test). Graph represents combined data from three independent experiments