Research ArticleInflammation

Manganese activates NLRP3 inflammasome signaling and propagates exosomal release of ASC in microglial cells

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Sci. Signal.  08 Jan 2019:
Vol. 12, Issue 563, eaat9900
DOI: 10.1126/scisignal.aat9900
  • Fig. 1 Manganese treatment induced NLRP3 inflammasome activation in microglial cells in vitro.

    (A) Western blot analysis of caspase-1 cleavage and IL-1β maturation in lysates from an LPS-primed microglial cell line treated with Mn, as indicated. Blots (left) are representative of four independent experiments. Normalized band intensity data (right) are means ± SEM from all experiments. Con, control. (B) Luminex analysis of IL-1β production by a microglial cell line primed with LPS and treated for 24 hours with Mn at the indicated dose. Data are means ± SEM pooled from eight independent experiments. (C) Immunofluorescence microscopy analysis of ASC spec formation in a microglial cell line primed with LPS and treated as indicated. Images (left) are representative of three independent experiments. Quantified data (right) are means ± SEM from all experiments. Scale bar, 20 μm. (D and E) Western blot (D) and immunofluorescence microscopy (E) analyses of NLRP3 abundance in a microglial cell line primed with LPS and treated as indicated. Blots and images are representative of three independent experiments. Normalized band intensity data (D, bottom) are means ± SEM from all experiments. Scale bar, 20 μm. (F and G) qRT-PCR analysis of IL-1β (F) and NLRP3, NLRC4, and AIM2 (G) mRNA expression after treatment of an LPS-primed microglial cell line. Data are means ± SEM pooled from three independent experiments. *P < 0.05, ***P < 0.001 by analysis of variance (ANOVA) with Tukey post hoc analysis.

  • Fig. 2 Manganese exposure induced NLRP3 inflammasome activation in microglial cells in vivo.

    (A) Western blot analysis of the NLRP3 and iNOS expression in wild-type microglial cells treated with Mn and αSynAgg as indicated. Blots (left) are representative of three independent experiments. Normalized band intensity data (right) are means ± SEM from all experiments. (B) Luminex analysis of IL-1β production by WT microglial cells treated with Mn and αSynAgg as indicated. Data are means ± SEM pooled from four independent experiments. (C) qRT-PCR analysis of NLRP3, NLRC4, and AIM2 mRNA expression in the striata of C57BL mice exposed to Mn for 30 days. Data are means ± SEM pooled from five mice from three experiments. (D) Western blot analysis of caspase-1 cleavage and IL-1β maturation in lysates from striatum samples from mice treated as indicated. Blots (top) are representative of six mice from three experiments. Normalized band intensity data (bottom) are means ± SEM from all experiments. (E) Immunofluorescence microscopy analysis of NLRP3 abundance in IBA1-positive microglial cells in the striatal region of mice treated as indicated. Images are representative of three mice from three experiments. Scale bar, 15 μm. *P < 0.05 and ***P < 0.001 by ANOVA with Tukey post hoc analysis.

  • Fig. 3 Mn treatment induced mitochondrial damage by modulating Mfn2 and Mul1 expression in microglial cells.

    (A to E) Seahorse analysis of the OCR in Mn-exposed LPS-primed primary microglial cells. Data were analyzed to extrapolate mitochondrial ATP production (B), basal respiration rate (C), maximal respiration (D), and spare respiratory capacity (E). Data are means ± SEM pooled from five independent experiments. (F) Immunofluorescence microscopy analysis of mitochondrial superoxide generation from microglial cells primed with LPS and exposed to Mn. Images are representative of three independent experiments. Scale bar, 100 μm. (G) qRT-PCR analysis of Mfn2 mRNA expression in Mn-exposed microglial cells. Data are means ± SEM pooled from three independent experiments. (H and I) Immunofluorescence microscopy (H) and Western blot analysis (I) of Mfn2 abundance in microglial cells primed with LPS and exposed to Mn. Images and blots are representative of four experiments. Normalized band intensity data (I, bottom) are means ± SEM from all experiments. Scale bar, 100 μm. (J) qRT-PCR analysis of Mul1 mRNA expression in Mn-exposed microglial cells. Data are means ± SEM pooled from three independent experiments. (K) Immunofluorescence microscopy analysis measuring Mul1 protein on Mn exposure. Images are representative of three independent experiments. Scale bar, 20 μm. *P < 0.05 and ***P < 0.001 by ANOVA with Tukey post hoc analysis.

  • Fig. 4 VPS35, a retromer complex protein, can modulate Mfn2 degradation during Mn-induced inflammasome activation.

    (A and B) qRT-PCR analysis of the retromer complex components VPS35 and VPS29 mRNA expression. Data are means ± SEM pooled from three independent experiments. (C and D) Immunofluorescence microscopy (C) and Western blot (D) analysis of VPS35 abundance in microglial cells exposed to Mn. Images and blots are representative of three independent experiments. Normalized band intensity data (D, bottom) are means ± SEM from all experiments. Scale bar, 20 μm. (E) qRT-PCR analysis of VPS35 mRNA expression in the striatum of mice exposed to Mn. Data are means ± SEM pooled from six mice from three independent experiments. (F) Western blot analysis of Mfn2 abundance in lysates from wild-type and VPS35 CRISPR-Cas9 KD microglial cells. Blots are representative of four independent experiments. Normalized band intensity data (D, bottom) are means ± SEM from all experiments. (G) qRT-PCR analysis of Mul1 mRNA expression in VPS35 KD microglial cells compared to wild-type cells. Data are means ± SEM pooled from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 by ANOVA with Tukey post hoc analysis.

  • Fig. 5 Exosomal release of the inflammasome component ASC propagates Mn-induced inflammasome activation.

    (A and B) qRT-PCR analysis of pro–IL-1β (A) and NLRP3 (B) mRNA expression in wild-type (WT) and Cav1-KD microglial cells after Mn treatment. Data are means ± SEM pooled from three independent experiments. (C) Luminex analysis of IL-1β secreted by wild-type and Cav1-KD or Cltc-KD microglial cells after Mn treatment. Data are means ± SEM pooled from eight independent experiments. (D) Western blot analysis of ASC abundance in lysates of exosomes (exo) isolated from Mn-treated microglial cells treated as indicated. The blot (left) is representative of three independent experiments. Normalized band intensity data (right) are means ± SEM from all experiments. (E) NanoSight analysis of the number of exosomes released from wild-type and ASC KO microglial cells. Data are means ± SEM from three independent experiments. (F) Immunofluorescence microscopy analysis of CFP expression in wild-type microglial cells after treatment with exosomes isolated from cells overexpressing ASC-CFP. Images (left) are representative of three independent experiments. Quantified data (right) are means ± SEM from all experiments. Scale bar, 5 μm. (G and H) qRT-PCR analysis of NLRP3 (G) and pro–IL-1β (H) mRNA expression in microglial cells after treatment with exosomes isolated from wild-type and ASC KO microglial cells. Data are means ± SEM pooled from three independent experiments. (I) Luminex analysis of IL-1β secreted by microglial cells treated with exosomes isolated from the serum of Mn-exposed animals. Data are means ± SEM pooled from eight independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 by ANOVA with Tukey post hoc analysis.

  • Fig. 6 Serum exosomes and serum from a welder population have a higher load of ASC and higher proinflammatory cytokines, respectively, when compared to age-matched controls.

    (A and B) Western blot analysis of exosomal ASC load in welders and age-matched controls. Blot (A) is representative of 17 controls and 20 welder samples. Normalized band intensity data (B) are means ± SEM from all experiments. (C to F) Luminex analysis of TNF-α (C), IL-17 (D), RANTES (E), and MIP1b (F) abundance in control and welder serum. Data are means ± SEM from 17 controls and 20 welder samples. (G and H) qRT-PCR analysis of pro–IL-1β (G) and NLRP3 (H) mRNA expression in microglial cells treated with exosomes isolated from welders or age-matched controls. Data are means ± SEM from five welders and four age-matched controls. *P < 0.05 by Student’s t test.

  • Fig. 7 Mn may induce mitochondrial dysfunction leading to inflammasome activation by degradation of Mfn2.

    Mn may also stimulate the release of exosomal ASC, thereby leading to NLRP3 inflammasome propagation. TLR, Toll-like receptor; IκB, inhibitor of NFκB.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/12/563/eaat9900/DC1

    Fig. S1. Mn specifically induced inflammasome activation in primary microglial culture.

    Fig. S2. Mn enhanced LPS-induced nitrite generation.

    Fig. S3. Mn exposure did not inhibit αSynAgg-induced proinflammatory cytokine production.

    Fig. S4. Caspase-1-p20–induced cleavage of pro–IL-1β.

    Fig. S5. Mn exposure inhibited microglial mitochondrial respiration.

    Fig. S6. LPS increased Mn transporters and Mn uptake.

    Fig. S7. Mn increased Mul1 expression in a dose-dependent manner.

    Fig. S8. CRISPR-Cas9 VPS35 KD results in Mfn2 degradation.

    Fig. S9. Caveolin and clathrin KD reduced Mn-induced Nos2 expression but not TNF-α.

    Fig. S10. Mn exposure did not change the size distribution of the exosomes secreted by microglial cells.

    Fig. S11. Exposure to exosomes isolated from Mn-gavaged animals did not alter release of IL-6 and TNF-α.

    Fig. S12. Mn concentration in whole blood is unaltered in welders.

    Fig. S13. Proinflammatory cytokine profile of welder and age-matched control populations.

  • This PDF file includes:

    • Fig. S1. Mn specifically induced inflammasome activation in primary microglial culture.
    • Fig. S2. Mn enhanced LPS-induced nitrite generation.
    • Fig. S3. Mn exposure did not inhibit αSynAgg-induced proinflammatory cytokine production.
    • Fig. S4. Caspase-1-p20–induced cleavage of pro–IL-1β.
    • Fig. S5. Mn exposure inhibited microglial mitochondrial respiration.
    • Fig. S6. LPS increased Mn transporters and Mn uptake.
    • Fig. S7. Mn increased Mul1 expression in a dose-dependent manner.
    • Fig. S8. CRISPR-Cas9 VPS35 KD results in Mfn2 degradation.
    • Fig. S9. Caveolin and clathrin KD reduced Mn-induced Nos2 expression but not TNF-α.
    • Fig. S10. Mn exposure did not change the size distribution of the exosomes secreted by microglial cells.
    • Fig. S11. Exposure to exosomes isolated from Mn-gavaged animals did not alter release of IL-6 and TNF-α.
    • Fig. S12. Mn concentration in whole blood is unaltered in welders.
    • Fig. S13. Proinflammatory cytokine profile of welder and age-matched control populations.

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