Table 1.
Primer sequences list
Fig 1.
Inflammasome components NLRP3, ASC and caspase-1 are expressed in microglia, but not in astrocytes.
mRNA and/or protein expression of inflammasome components and substrates NLRP1, NLRP3, NLRC4, ASC (Pycard), Aim-2, Caspase-1, Caspase-4, IL-1α, IL-1β, IL-18 and HMGB1, were studied in mouse microglia, astrocytes, or bone-marrow derived macrophages (BMDM) after 6 hours of stimulation with LPS or Complete Cytokine Mix. RNA was extracted and analyzed for gene expression, relative to L27, by Real-Time PCR (A, C, D). Bar graphs shown represent the mean ± SD. (B) NLRP3, ASC and Caspase-1 expression were analyzed by Western Blot and α-Tubulin was used as loading control. (E) Microglia were analyzed by immunofluorescence for the expression of Iba1, NLRP3, ASC, Caspase-1, HMGB1 and IL-1β, nuclei are stained by DAPI, scale bar: 50μm. Data shown are the mean ± SD of at least three independent experiments. *p<0.05 compared to control (ctrl).
Fig 2.
NLRP3 inflammasome activation mechanism in microglia.
LPS primed microglia were stimulated with ATP (1 mM, 30 min), Nigericin (Nig, 1.34 μM, 2 h), Monosodium urate (MSU, 100 μg/ml, 5 h) or Aluminium hydroxide (Alum, 300 μg/ml, 5 h). IL-1β production and caspase-1 cleavage were assessed by Western blot (A). Secretion of IL-1β (B), IL-18 (C) and IL-1α(D) in supernatants of wild-type (WT), Nlrp3-/- and Casp1-/- microglia was assessed by ELISA. IL-1β secretion was assessed by ELISA in WT and P2rx7-/- microglia (E) and after treatment with different inhibitors (F-H) (± KCl (25 mM), N-acetyl cystein (NAC, 5 mM), Ca-074Me (10 μM) and cytochalasinD (cytoD, 2 μM)). Inhibitors were added for 30 min prior to inflammasome activation. Data shown are the mean ± SD of at least three independent experiments. *p<0.05, ns = not significant, compared to control (ctrl) (Fig 3B–3E) or compared to no treatment (Fig 3F–3H), #p<0.05, KO compared to WT.
Fig 3.
Amyloid-β25–35 activates microglia to secrete IL-1β independently from P2X7 signaling.
Untreated or LPS primed microglia were stimulated with amyloid-β (Aβ)25–35 (20 or 50 μM) or Aβ35–25 (50 μM) for 5 h. (A) RNA was analyzed for expression of Nlrp3, Il1b and Tnf, relative to L27, by Real-Time PCR (B) Cell free culture supernatants (SN) and cell lysates (XT) were analyzed by Western Blot for expression of caspase-1 and IL-1β. α-Tubulin was used as loading control. (C) IL-1β production in culture supernatants was assessed by ELISA. (D, E) IL-1β production in culture supernatants was assessed by ELISA in wild type, Nlrp3-/- and Casp1-/- (D), or P2rx7-/- (E) microglia. (F) ATP release was quantified in cell supernatant upon treatment. (G) Untreated or LPS primed microglia were stimulated with amyloid-β (Aβ) 1–42 (10μM) for 6h, 24h or 48h. IL-1β production in culture supernatants was assessed by ELISA. (H) Cell free culture supernatants (SN) and cell lysates (XT) were analyzed by Western Blot for expression IL-1β. α-Tubulin was used as loading control (I). Data shown are mean ± SD of at least three independent experiments. *p<0.05 compared to control (ctrl), #p<0.05, KO compared to WT.
Fig 4.
α-synuclein is not an NLRP3 inflammasome activator.
Microglia were stimulated with 5 μM α-synuclein (α-syn), either wild-type (α-syn WT) or mutant (α-syn A53T) (oligomeric form: olig. or fibrillated form: fib.) for 6 h. (A) RNA was extracted and analyzed for expression of Nlrp3, Il1b and Tnf, relative to L27, by Real-Time PCR. (B) After 6 h, 24 h or 48 h of stimulation with α-syn, or 6 h with LPS, cell lysates were analyzed by Western Blot for expression of pro-IL-1β and NLRP3. α-Tubulin was used as loading control. (C) LPS primed microglia were stimulated for 6 h with α-syn or for 30 min with ATP (1 mM) and IL-1β production in culture supernatants was assessed by ELISA. Data shown are the mean ± SD of at least three independent experiments. *p<0.05 compared to control (ctrl).
Fig 5.
Astrocytes are not able to secrete inflammasome substrates IL-1β and IL-18.
Astrocytes were treated for 6h with LPS, P3C, IL-1β, TNFα, IFNγ or CCM and RNA was extracted and analyzed for expression of Il6, Nos2, Cxcl10 (A), Il1b, Nlrp3 and IL18 (B) relative to L27, by Real-Time PCR. CXCL10 secretion was assessed after 6h or 24h of CCM treatment (C). Mouse astrocyte-enriched cultures (AEC-M2) and microglia were primed with Complete Cytokine Mix for (24h) or LPS (6h), respectively, prior to stimulation with ATP (1 mM, 30 min), Nig (1,34 μM, 2h), Aβ25–35 (20 μM, 5h), WT and mutant α-synuclein (5 μM, 5h) or poly(dA:dT) (1 or 2,5 μg/mL, 5h). Levels of IL-1β(D, E and F) and IL-18 (G) in culture supernatants were assessed by ELISA. Data shown are the mean ± SD of at least three independent experiments *p<0.05 compared to control (ctrl).