TLR-stimulated IRAKM activates caspase-8 inflammasome in microglia and promotes neuroinflammation
Cun-Jin Zhang, … , Richard M. Ransohoff, Xiaoxia Li
Cun-Jin Zhang, … , Richard M. Ransohoff, Xiaoxia Li
Published October 29, 2018
Citation Information: J Clin Invest. 2018;128(12):5399-5412. https://doi.org/10.1172/JCI121901.
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Research Article Autoimmunity Inflammation

TLR-stimulated IRAKM activates caspase-8 inflammasome in microglia and promotes neuroinflammation

Abstract

NLRP3 inflammasome plays a critical spatiotemporal role in the pathogenesis of experimental autoimmune encephalomyelitis (EAE). This study reports a mechanistic insight into noncanonical NLRP3 inflammasome activation in microglia for the effector stage of EAE. Microglia-specific deficiency of ASC (apoptosis-associated speck-like protein containing a C-terminal caspase-activation and recruitment [CARD] domain) attenuated T cell expansion and neutrophil recruitment during EAE pathogenesis. Mechanistically, TLR stimulation led to IRAKM–caspase-8–ASC complex formation, resulting in the activation of caspase-8 and IL-1β release in microglia. Noncanonical inflammasome-derived IL-1β produced by microglia in the CNS helped to expand the microglia population in an autocrine manner and amplified the production of inflammatory cytokines/chemokines. Furthermore, active caspase-8 was markedly increased in the microglia in the brain tissue from patients with multiple sclerosis. Taken together, our study suggests that microglia-derived IL-1β via noncanonical caspase-8–dependent inflammasome is necessary for microglia to exert their pathogenic role during CNS inflammation.

Authors

Cun-Jin Zhang, Meiling Jiang, Hao Zhou, Weiwei Liu, Chenhui Wang, Zizhen Kang, Bing Han, Quanri Zhang, Xing Chen, Jianxin Xiao, Amanda Fisher, William J. Kaiser, Masanori A. Murayama, Yoichiro Iwakura, Ji Gao, Julie Carman, Ashok Dongre, George Dubyak, Derek W. Abbott, Fu-Dong Shi, Richard M. Ransohoff, Xiaoxia Li

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Figure 2

Microglia processes IL-1β in an ASC-NLRP3–caspase-8–dependent manner.

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Microglia processes IL-1β in an ASC-NLRP3–caspase-8–dependent manner. (A...
(A and B) Primary microglia from 6-week-old mice were primed with LPS for 4 hours prior to stimulation with ATP for the indicated times. Cell lysate and supernatants were collected together and immunoblotted with the indicated antibodies. (C) Results of immunoblotting with antibodies to the indicated proteins for microglia stimulated with LPS (4 hours) plus ATP (30 minutes) and immunoprecipitated with anti-ASC. (D) Primary microglia from adult mice were primed with LPS for 4 hours prior to stimulation with ATP. Cell lysate and supernatants were collected together and immunoblotted with the indicated antibodies. (E) IL-1β ELISA of cell-free supernatants from adult mice–derived primary microglia treated with LPS for 4 hours and ATP for 15 or 30 minutes (n = 3/group). (F) IL-1β ELISA of primary microglia from adult mice treated with LPS and indicated inhibitors for 4 hours prior to stimulation with 0.2 mM ATP for the indicated times. UN, untreated; YVAD-fmk, caspase-1 inhibitor; IETD-fmk, caspase-8 inhibitor (n = 4/group). (G and H) FACS analysis of caspase-8–FLICA (fluorescent labeled inhibitors of caspases) in microglia of EAE mice at peak disease in vivo (n = 6/group). (I) Primary microglia from 6-week-old mice were stimulated with TLR agonists HMGB1 and CpGb for 8 hours prior to stimulation with 0.2 mM ATP. Cell lysate and supernatants were collected together and immunoblotted with the indicated antibodies. (J) Human microglia cell line SV40 was stimulated with LPS (100 pg/ml) for 4 hours and ATP (0.2 mM) for 30 or 60 minutes. Cell lysate and supernatants were collected together and immunoblotted with the indicated antibodies. (K) Clinical score for WT→Casp8fl/+Cx3cr1Cre-ER Ripk3–/– (Caspase8ΔWT) and WT Casp8fl/flCx3cr1Cre-ER Ripk3–/– (Caspase8Δmicroglia) bone marrow chimera mice (n = 6/group). Data are representative of 2 independent experiments; mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (unpaired 2-tailed Student’s t test). EAE clinical score by 2-way ANOVA.