Role of mitochondria ROS generation in ethanol-induced NLRP3 inflammasome activation and cell death in astroglial cells.

Chronic ethanol intake increases caspase-1 activity in cortical astroglial cells of TLR4 mice. (A–D) Confocal images illustrate the co-localization of caspase-1 (red) with GFAP (green) in the cortex area (E) of TLR4+/+ (A,B) and TLR4-/- (C,D) mice (arrows indicate co-localization of the Casp-1 and GFAP-positive cells). (F) Confocal negative controls, in the absence of primary antibodies. We used four independent biological replicates from each experimental condition. Scale bar: 75 μm. (G) The quantitative analysis shows the percentage of the increased number of Casp-1/GFAP-positive co-localized cells in the cortices of ethanol-treated WT mice in relation to the untreated control mice. Non-significant changes were observed for the treated/untreated TLR4-/- (TLR4-KO) mice. (H) Caspase-1 enzymatic activity was determined in the brain cortex. (I,J) The analysis of the IL-1β and IL-18 cytokines in the cortical homogenates of TLR4 mice was conducted by ELISA. Values represent the [mean ± SEM] of at least six to eight individual experiments.*p < 0.05, **p < 0.01 (Mann–Whitney U non-parametric test or a Student’s t-test).

Astrocytes in primary culture expresses high levels of NLRP3 mRNA. (A) mRNA expression of NLRP3, AIM2, NLRP1, and IPAF inflammasomes in cultured astrocytes evaluated by RT-PCR. (B) Ethanol treatment (10 and 50 mM) for 24 h up-regulates the NLRP3 mRNA levels in the cultured astrocytes from TLR4 mice. We used n = 5–7 independent experiments and ratios were normalized with the PPIA housekeeping gene. Bars represent the [mean ±SEM]. *p < 0.05, **p < 0.01, ***p < 0.001 (Mann–Whitney U non-parametric test or a Student’s t-test). (C) Immuno-fluorescence of NLRP3 (green) and GFAP- (red) co-localization in the astrocytes from TLR4 and TLR4-knockout mice. Scale bar 20 μm.

Ethanol treatment activates the NLRP3 inflammasome complex in cultured astrocytes from TLR4-WT mice. (A) Western blot analysis shows the NLRP3 and p10/caspase-1 active cleavage (the initiator caspase in pyroptosis) protein levels in the LPS-, ATP-, and ethanol- (10 and 50 mM) treated astrocytes. (B) We show ASC oligomerization and quantification of the ASC dimer by densitometry. Cells were lysed, pelleted by centrifugation and incubated with DSS for 30 min. The cross-linked pellets were resuspended in SDS sample buffer, and proteins were separated using 12% SDS-PAGE and Western blotted with anti-mouse ASC antibodies as described under Section “Material and Methods.” The presence of dimers and trimers was observed in the ATP-, LPS-, or ethanol-treated astrocytes correlating with a significative up-regulation of ASC dimers. (C) Cell lysates were collected and co-immunoprecipitated with the NLRP3 Ab (IP), and the immune complexes were detected by Western blot with Caspase-1 (IB). We show the presence of the p45 caspase-1 precursor and the active p20/p10 Caspase-1 in treated/untreated astrocytes. The mouse IgG was used as a negative control. (D) ELISA measured determined the IL-1β and IL-18 levels in the supernatant of the astrocytes treated with LPS, ATP and ethanol (10 and 50 mM) after 24 h. Non-significant differences were observed between the treated or non-treated TLR4-KO astrocytes. Values represent the mean ± SEM of 3–9 individual experiments. #p < 0.06, *p < 0.05, **p < 0.01, ***p < 0.001 (Mann–Whitney U non-parametric test or a Student’s t-test).

The ethanol-induced mitochondria ROS production level measured by MitoSOX Red fluorescence intensity. (A) A flow cytometry analysis shows that the ATP or LPS or ethanol (10 and 50 mM) treatments increased mROS generation in the WT astrocytes. Incubation with Z-YVAD-FMK, Z-VAD-FMK, the NLRP3 blocking peptide or Mito-TEMPO before and during treatments notably reduces mROS generation activation in WT astrocytes. (B) No significant changes in mROS generation were observed in the TLR4-KO-astrocytes incubated with the same inhibitors and treatments as used in the TLR4-astrocytes. Bars represent the (mean ± SEM) of 4–12 individual experiments. *p < 0.05, **p < 0.01, ***p < 0.001 (one-way ANOVA followed by a Dunnett’s Multiple Comparison Test or a Student’s t-test).

Ethanol enhances the production of IL-18 and IL-1β in WT astroglial cells. (A,B) Graphs represent the levels of IL-18 (A) and IL-1β (B) measured in the supernatant medium of the WT and TLR4-KO astrocytes treated with ATP, LPS or ethanol (10 and 50 mM) for 24 h. Incubation with Z-YVAD-FMK, Z-VAD-FMK, the NLRP3 blocking peptide or Mito-TEMPO, along with the different treatments, reduces, or even abolishes, the up-regulation of the cytokine release. No changes in the levels of cytokines were noted in the supernatant medium for the TLR4-KO-astrocytes for any of the treatments and inhibitors used, except for the Mito-TEMPO treatment at the IL-18 released levels. Bars represent the (mean ± SEM) expressed as a percentage (%) of 6–10 individual experiments. *p < 0.05, **p < 0.01, ***p < 0.001 (two-way ANOVA with post hoc Bonferroni’s correction).

Confocal images of NLRP3/caspase-1 co-localization within mitochondria of the astrocytes treated with ethanol, ATP, or LPS. Microphotographs show that the ATP, LPS, or ethanol (10 mM) treatments promote the co-localization of NLRP3 inflammasome (blue) with active caspase-1 (green) within mitochondria (red) in the WT-astrocytes when compared with the untreated control astrocytes (A). Astrocytes treated with Z-VAD-FMK (B) and with Mito-TEMPO (C) treatments do not induce caspase-1 activation in both WT and TLR4-KO astrocytes. We performed at least three independent experiments under each experimental condition. Representative pictures are presented.

Activation of the NLRP3 inflammasome complex triggers pyroptosis and apoptosis in ethanol-induced astroglial cells. (A) Bars show the percentage of cell dead by pyroptosis, necrosis and apoptosis in astrocytes treated for 24 h with ATP, LPS, and ethanol (10 and 50 mM) evaluated by an In Cell Analyzer. Approximately 1000–5000 cells were analyzed/experimental condition. Bars represent the (mean ± SD). (B) LDH activity in the supernatant astrocytes was measured with different treatments. Bars represent the (mean ± SEM) of at least 6–10 individual experiments. *p < 0.05, **p < 0.01 (Mann–Whitney U non-parametric test).

Ethanol can also induce the apoptosome formation in astrocytes. (A) The Western blot analyses of Apaf-1, the active fragments from caspase-9 (37 kDa) and caspase-3 (17 kDa), in the astrocytes treated with ATP (5 μM) or ethanol (10 and 50 mM). Bars represent the (mean ± SEM) of at least 6–10 individual experiments. *p < 0.05, **p < 0.01 (Mann–Whitney U non-parametric test or a Student’s t-test). (B) The percentage of apoptosis in the astrocytes incubated with ATP or ethanol (10 mM, 50 mM) for 24 h was assessed by a TUNEL assay in the WT and TLR4-KO astrocytes. Scale bar: 20 μm. Arrowheads show different apoptotic processes, blebbing and nuclei condensation. Asterisk show positive TUNEL cells. Bars represent the (mean ± SEM) of three individual experiments. *p < 0.05, **p < 0.01 (two-way ANOVA with post hoc Bonferroni’s correction).