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. 2018 Dec 3;128(12):5399-5412.
doi: 10.1172/JCI121901. Epub 2018 Oct 29.

TLR-stimulated IRAKM Activates caspase-8 Inflammasome in Microglia and Promotes Neuroinflammation

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Free PMC article

TLR-stimulated IRAKM Activates caspase-8 Inflammasome in Microglia and Promotes Neuroinflammation

Cun-Jin Zhang et al. J Clin Invest. .
Free PMC article

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.

Keywords: Autoimmune diseases; Autoimmunity; Inflammation.

Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1
Figure 1. Microglia-specific ASC deficiency attenuated EAE disease.
Analysis of results for WT→ASCfl/+Cx3cr1Cre-ER (ASCΔWT) and WT→ASCfl/flCx3cr1Cre-ER (ASCΔmicroglia) bone marrow chimera mice in EAE disease. (A) FACS analysis of CreER-EYFP expression in microglia of ASCΔWT mice with or without tamoxifen administration on day 16 of EAE induced by active immunization with MOG35–55. (B) Mean clinical score for EAE in ASCΔWT (n = 6) and ASCΔmicroglia (n = 5) bone marrow chimera mice induced by active immunization with MOG35–55. Absolute numbers (C) and gating strategy (D) of immune cell infiltration determined at the peak of disease in brains of EAE mice by flow cytometry (n = 3/group). (E) Inflammatory gene expression in the lumbar spinal cords as assessed at the peak of disease (n = 4). (F) Luxol Fast Blue and H&E staining of lumbar spinal cords harvested at the peak of disease. Scale bars: 200 μm. (G and H) Mean clinical score for EAE in ASCΔWT and ASCΔmicroglia bone marrow chimera mice induced by adoptive Th17 (G) (n = 7 and n = 5, respectively) or (H) Th1 (n = 5/group) transfer. Data are representative of 2 independent experiments; mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 (unpaired 2-tailed Student’s t test). EAE clinical score by 2-way ANOVA.
Figure 2
Figure 2. Microglia processes IL-1β in an ASC-NLRP3–caspase-8–dependent manner.
(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.
Figure 3
Figure 3. IRAKM controls the activity of caspase-8 and IL-1β production in microglia.
(AC) Results of immunoblotting with antibodies to the indicated proteins for primary microglia stimulated with LPS (4 hours) plus ATP (30 minutes) and immunoprecipitated with anti–caspase-8 (A), anti-IRAKM (B), or anti-ASC (C). (D) Primary microglia from 6-week-old mice with indicated genotyping were primed with LPS (100 pg/ml) for 4 hours prior to stimulation with 0.5 mM ATP for the indicated times. Cell lysate and supernatants were collected together and immunoblotted with the indicated antibodies. (E) Primary microglia from 6-week-old mice with indicated genotypes was stimulated with LPS (0.1 μg/ml, 4 hours) plus ATP (30 minutes) and stained with caspase-8–FLICA in the last hour, followed by flow analysis of caspase-8 activation (n = 4/group). (F) IL-1β ELISA of cell-free supernatants from adult mice–derived primary microglia treated with LPS (100 pg/ml) for 4 hours and ATP (0.2 mM) for 15 or 30 minutes (n = 4/group). (G) Primary microglia from mice with indicated genotypes was primed with LPS (100 pg/ml) for 4 hours prior to stimulation with 0.2 mM ATP for the indicated times. Cell lysate and supernatants were collected together and immunoblotted with the indicated antibodies. (HJ) Results of immunoblotting with antibodies to the indicated proteins for primary microglia stimulated with LPS (100 pg/ml, 4 hours) plus ATP (30 minutes) and immunoprecipitated with anti-ASC (H) or anti-IRAKM (I and J). Data are representative of 2 independent experiments; mean ± SEM. *P < 0.05 (unpaired 2-tailed Student’s t test).
Figure 4
Figure 4. IRAKM controls the activity of caspase-8 and IL-1β production in microglia in vivo.
(A and B) Targeting vector design for generation of a mouse strain with Irak3 exon 3 flanked by loxp sites (A), and Western blot analysis of IRAKM expression in FACS-sorted microglia from indicated mice (B). (C) Mean clinical score for EAE in WT→Cx3cr1creIrakmfl/+ (IRAKMWT, n = 5) and WT→Cx3cr1creIrakmfl/fl (IRAKMΔmicroglia, n = 7) bone marrow chimera mice induced by active immunization with MOG35–55. (D) Absolute numbers of immune cell infiltration as well as resident microglia determined at the peak of disease in brains of EAE mice by flow cytometry (n = 3/group). (E) H&E and Luxol Fast Blue staining of lumbar spinal cords harvested at the peak of disease. Scale bars: 200 μm. (F) Flow cytometry analysis of caspase-8 activation in microglia of EAE mice at peak disease (n = 4). (G) Primary microglia of EAE brains were isolated and cultured for 24 hours ex vivo. Cell-free supernatant was collected for IL-1β ELISA (n = 4). Data are representative of 2 independent experiments; mean ± SEM. *P < 0.05, **P < 0.01 (unpaired 2-tailed Student’s t test). EAE clinical score by 2-way ANOVA.
Figure 5
Figure 5. Dynamics of caspase-8 activation, IL-1β production, and IL-1R expression in microglia.
(A and B) Analysis of results for WT→ASCfl/+Cx3cr1Cre-ER (ASCΔWT) bone marrow chimeric mice in EAE disease. Flow cytometry analysis of activated caspase-8 in primary EYFP+ microglia isolated from brains of EAE mice at the indicated time points (n = 4). The quantification of activated caspase8+ microglia is shown in B. (C and D) Brain specimens of 7 controls and 11 MS cases were provided by the Multiple Sclerosis Tissue Bank at Imperial College London. The edges of cortical active lesions in brain slices were stained with the indicated antibodies against microglia markers TMEM119 and activated caspase-8–FLICA. Scale bars: 50 μm (C). The activated caspase-8+ cells were quantified (D). (E) EFYP+ primary microglia sorted from EAE mice (ASCΔWT) at the indicated times was cultured overnight and supernatant was subjected to ELISA analysis of IL-1β production (n = 4). (F) FACS analysis of IL-1R expression in EFYP+ primary microglia isolated from brains of WT naive and EAE mice (ASCΔWT) (n = 4). 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).
Figure 6
Figure 6. Microglia-intrinsic caspase-8 inflammasome activation is required for microglia survival and proliferation in the CNS.
(A) The proliferation of microglia from WT EAE mouse brain at peak disease was analyzed by flow cytometry after staining with antibodies against CD45, CD11b, IL-1R, Ki67, and caspase-8–FLICA. (B) Primary microglia isolated from mouse brain with indicated genotypes were stimulated with LPS+ATP in the presence of IL-1Rα followed by PI staining and flow cytometry analysis (n = 3). (C and D) Spinal cord from EAE mice with indicated genotypes at peak disease were stained with antibodies against caspase-8–FLICA, Ki67, and Brdu. EYFP indicates the microglia (n = 6/group). WT→Cx3cr1Cre-ER (Cx3Cr1ΔWT). Scale bars, 50 μm. (E) Flow cytometry analysis of Ki67+ microglia in brains of EAE mice with indicated genotypes (n = 6). (FJ) WT and Il1b–/– microglia isolated from adult mouse brain were transferred to Csf1rfl/flCx3cr1cre mice after the fourth tamoxifen injection (5 mg/mouse/week, i.p.) and the mice were subjected to EAE induction 2 weeks after the last tamoxifen injection (n = 6). Clinical score was presented as mean ± SEM (F). Total microglia (G) and Ki67+ microglia (H) in brains at EAE peak were analyzed by flow cytometry. Brdu+ microglia were analyzed in brain slices (I and J). Scale bar, 50 μm. Data are representative of 2 independent experiments; mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 (unpaired 2-tailed Student’s t test). EAE clinical score by 2-way ANOVA.
Figure 7
Figure 7. Microglia-intrinsic IRAKM–caspase-8 inflammasome activation is required for neutrophil recruitment and T cell expansion in the CNS.
(A) Microglia were isolated from brains of EAE mice with indicated genotypes at peak disease and cultured for 24 hours. The supernatant was analyzed by ELISA for cytokine and chemokine production. (BD) Flow cytometry analysis of microglia (B), IL-17+CD4+, IFN-γ+CD4+ cell (C) and neutrophil (D) in brains of EAE mice with indicated genotypes at different time points after active immunization with MOG35–55. (E) IL-17+CD4+, IFN-γ+CD4+ cells, and neutrophil in brains of mice indicated in Figure 6F were analyzed by flow cytometry. Data are representative of 2 independent experiments (n = 4/group). Mean ± SEM. *P < 0.05, **P < 0.01 (unpaired 2-tailed Student’s t test).
Figure 8
Figure 8. Model of microglia-intrinsic IRAKM–caspase-8 noncanonical inflammasome pathway in EAE/MS pathogenesis.

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