Ethanol-mediated activation of the NLRP3 inflammasome in iPS cells and iPS cells-derived neural progenitor cells.

Ethanol activates the inflammasome pathway in iPS cells. iPS cells were cultured in 70 mM ethanol for 24hr or 7d. a From the top: Immunofluorescence analysis by confocal microscopy shows the expression of pluripotency markers Oct3-4, Tra-1-60, Sox2, and of the proliferation marker Ki-67 in iPS cells after 24hr or 7d ethanol exposure. Scale bar: 50 μm. b Growth curve of iPS cell lines #1 and #2. Inserts: showing the doubling time (days in vitro) (left), and the relative percentage of pyknotic nuclei over total DAPI+ nuclei (right). c Confocal microscopy images showing the expression of the apoptotic marker, Cleaved Caspase-3 (Casp3) and of the inflammasome-related markers, Caspase-1 (Casp1) and NLRP3. Scale bars: 50 μm. d Western Blot analysis and graph showing the relative densitometric analysis of expression of the inflammasome-pathway markers Casp1 (p45 and p10) and NLRP3. Quantification of the proteins was normalized to β-actin expression. e Graph showing the relative percentage of Casp3+ cells over the total number of DAPI+ cell nuclei. f Western Blot analysis of Casp3 expression

Ethanol activates the inflammasome pathway in NPCs. NPCs were obtained from iPS cells by epigenetic neural induction and treated with ethanol for 24hr or 7d. a From the top: phase contrast pictures show that the cell density between the different treatments is comparable at day 7. Scale bar: 200 μm. Immunofluorescence analysis by confocal microscopy shows the expression of neural multipotency markers Nestin, Pax6, and Sox2 in NPCs. Scale bar: 50 μm. b Graphs showing the relative percentage of Nestin+, Pax6+ and Sox2+ cells over the total number of DAPI+ nuclei. c Fluorescent images of EdU+ nuclei after incorporation assay for 24hr at day 7 of ethanol exposure. Scale bar: 50 μm. d Graph showing the relative percentage of EdU+ cells over the total number of DAPI+ nuclei. e Growth curve of NPC lines #1 and #2 after acute or chronic ethanol exposure. f Immunofluorescence analysis by confocal microscopy shows the expression of the inflammasome markers Casp1 and NLRP3, autophagy marker LC3B, and of the apoptotic marker Casp3 in NPCs with or without ethanol exposure (24hr or 7d). Scale bar: 20 & 50 μm (LC3B). g Graph showing the relative percentage of Casp1+ cells over the total number of DAPI+ nuclei. h Western Blot and graph showing the relative densitometric analysis of Casp1 (p45 and p10) expression. Quantification of relative protein expression normalized to VCP expression. i Western Blot and graph showing the relative densitometric analysis of NLRP3 expression. Protein quantification was normalized to β-actin. j Graph showing the relative percentage of LC3B puncta per cell. k Graph showing the relative percentage of Casp3+ cells over the total number of DAPI+ nuclei. The differences among all the values were not statistically significant unless indicated (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001). Student’s t-test was utilized for all experiments

Early ethanol exposure leads to a decrease of NPC-derived neurons. NPCs were pretreated with ethanol for 24hr or 7d and differentiated to neurons for 26 days. a Phase contrast pictures of treated and untreated NPCs after 7 days of differentiation toward the neuronal lineage, and immunofluorescence analysis of neurons differentiated from NPCs showing the synaptic marker synapsin, and glutamatergic marker vGlut. Scale bars: 200 μm (top), 10 μm (middle, bottom). b Graph showing the relative percentage of MAP2+ cells over the total number of DAPI+ nuclei. c Number of synapses quantified by synapsin+ puncta per 100 μm dendrite length under control or ethanol pre-exposure conditions. d Electrophysiological analysis. e Immunofluorescence analysis showing the expression of Casp1, MAP2, and NLRP3. Scale bar: 50 μm. The differences among all the values were not statistically significant unless indicated (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001). Student’s t-test was utilized for all experiments

Ethanol alters mitochondrial patterns in iPS cells, NPCs, and NPC-derived neurons. Confocal microscopy images showing the mitochondrial pattern (Mitotracker) in iPS cells (a), NPCs (b), and NPC-derived neurons (after 26 days of differentiation) (c) after treatment with ethanol for 24hr or 7d. Nuclei (in blue) are counterstained with DAPI. Inserts: co-immunolabeling with MAP2 shows different colocalization of Mitotracker in neuronal cells. Scale bars: 10 μm

Ethanol alters lysosomal patterns in iPS cells, NPCs, and NPC-derived neurons. Analysis of co-localization of the lysosomal marker Lamp1 with Mitotracker in iPS cells (a) and NPCs (b) after 24hr or 7d treatment with ethanol, and co-localization of Lamp1 with β-tubulinIII in untreated and treated NPC-derived neurons (c). Scale bars: 10 μm (a), 20 μm (b and c)

Cooperative effects of ethanol and peroxide challenges on apoptosis and lysosomal/mitochondrial distribution. At day 7 after exposure to ethanol for 24hr or 7d, iPS cells were exposed for 14hr to 5 or 10 μM H2O2 and immunostained with antibodies against Oct4 and Casp3 (a), or stained with Mitotracker™ and Lamp1 (b). Both single treatments with ethanol and H2O2 alter the normal patterns of the cell, but a remarkable enhancement of the effects observed following a single challenge is evident following a double challenge. Scale bars: 50 μm (a), 10 μm (b)

Cooperative effects of ethanol and peroxide challenges on inflammasome markers NLRP3 and Casp1 in iPS cells. On day 7 following 24hr or 7d ethanol treatment, iPS cells were exposed for 14hr to 5 or 10μM H2O2 and immunostained with antibodies against NLRP3 and Casp1 (a), and LC3B (b). At 10μM H2O2, iPS cells pretreated with ethanol for 7d displayed a dramatic increase in death. Scale bars: 50 μm (a), 10μm (b)

Cooperative effects of ethanol and peroxide challenges on Casp3+ cells and LC3B puncta in iPS cells. a Graph showing the relative percentages of Casp3+ cells over the total number of DAPI+ nuclei. Low panels: Statistical significance was indicated. b Graph showing the relative percentages of LC3B puncta per cell. Low panels: Statistical significance was indicated. Student’s t-test was utilized for all experiments

Cooperative effects of ethanol and peroxide challenges in NPCs. At day 7 after exposure to ethanol for 24hr or 7d, NPCs were exposed for 14hr to 100 or 500 μM H2O2 and immunostained with antibodies against Oct4 and Casp3 (a), NLRP3 and Casp1 (c), and LC3B (d), or stained with Mitotracker and Lamp1 (b). Both single treatments with ethanol and H2O2 alter the normal patterns of the cell, but a remarkable enhancement of the single effects is evident by the concurrence of the two treatments. Scale bars: 50 μm (a–c), 10 μm (b–d)