Fig 2: Mature OLs undergo GSDMD-marked pyroptotic injury and inflammation.(A) Postmortem cortex brain sections from normal (Nor) subjects and AD patients (Table 1) were stained with anti-MBP, anti-GSDMD, and anti–6E-10 antibodies. (B) Quantification: (top) colocalization of GSDMD and Aß plaques; (middle) localization of GSDMD in MBP+ OLs; (bottom) the area covered by MBP+ OLs in Aß plaques and non-Aß plaque area. Student’s t test. (C) Postmortem cortex brain sections from normal subjects and AD patients were stained with anti-CNPase, anti-GSDMD, and anti–6E-10 antibodies. (D) Quantification: (top) the colocalization of GSDMD in Aß plaques; (middle) the localization of GSDMD in CNPase+ OLs; (bottom) the area covered by CNPase+ OLs in Aß plaques and non-Aß plaque area. Student’s t test. Note: To quantify the colocalization of proteins, Pearson’s correlation coefficient of two proteins per 45,000 µm2 was determined using ImageJ plug-in JaCoP. Fifteen to 21 images per group were analyzed. The brains of WT and 5XFAD mice were harvested at the ages of 3, 6, and 9 months (M). Brain sections were stained with (E) anti-GSDMD (green) and anti-CC1 (a marker of mature OL, red) antibodies, (F) anti–IL-1ß (green) and anti-CC1 (red), and (G) anti-neurofilament (NF, green) and anti-MBP (red). n = 3 to 5 mice per group. The histogram shows the intensities of GSDMD (H) and IL-1ß (I) in the CC1+ OLs, and the intensities of MBP (J) and NF (K) in the corpus callosum of mice. Scale bar, 20 µm. The data were compared by one-way analysis of variance (ANOVA) with Tukey’s post hoc test. All data are expressed as the means ± SEM.

Fig 3: NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3)-dependent GSDMD cleavage provokes pyroptosis in PRRSV infection. (A) Cell lysates were prepared and the pellets were subjected to ASC (apoptosis-associated speck-like protein containing a CARD) oligomerization assays (top panel). ASC and PRRSV-N in the supernatants were determined by Western blotting, with GAPDH as an internal control of the lysate supernatants (bottom row). (B) Representative images of ASC immunohistochemical staining in lung sections from 6-week-old pigs. Arrows indicate ASC specks. (C) PAMs were infected with PRRSV (MOI = 1) at the indicated times or administered LPS for 6 h followed by nigericin for 1 h as a positive control. Anti-ASC antibody or isotype control IgG was used for immunoprecipitation, followed by immunoblotting for NLRP3, CASP1, and ASC. Results are representative of two individual experiments. (D) A representative image of Marc-145 cells containing NLRP3 aggregates (green). Marc-145 cells were infected with PRRSV (MOI = 1) for 24 h or treated with LPS for 12 h followed by nigericin for 1 h as a positive control. Subcellular localizations of NLRP3 (green) and dsRNA (red) and the nucleus marker DAPI (blue) were examined under confocal microscopy. Inset (white) shows a higher magnification of a cell containing NLRP3 puncta. (E) Representative images of NLRP3 immunohistochemical staining in lung sections from 6-week-old pigs. Arrows indicate NLRP3 aggregates. (F) PAMs were pretreated with MCC950 for 1h before being infected with PRRSV (MOI = 1) for 24 h or stimulated with LPS and nigericin. Cleaved GSDMD and activated CASP1 in cell lysates were analyzed by Western blotting using the indicated antibodies. GAPDH is shown as an internal control. MCC950 concentrations of 5 and 20 µM are indicated by “+” and “++,” respectively. Data are representative of the results of three independent experiments.

Fig 4: LDC7559 and NA-11 inhibit NETosis independent of GSDMD but elicit identical phenotypes in neutrophils(A and B) LDH (A) or IL-1ß (B) released from primary human monocytes. Bars show the mean ± SEM of monocytes from three donors.(C) Western blots of monocytes at 1 h after electroporation with LPS. FL, full length GSDMD; NT, N-terminal fragment of GSDMD. Results representative of three independent experiments.(D) Coomassie blue staining of recombinant GSDMD. Results representative of three independent experiments.(E) TR-FRET assay measuring Europium-labeled biotin released from liposomes exposed to caspase-4 and GSDMD. Symbols indicate the mean ± SD of three independent experiments.(F and G) Percentage of polymorphonuclear leukocytes (PMNs) undergoing NETosis induced by PMA (F) or the stimuli indicated (G). Data are the mean ± SD of cells from three donors.(H) ROS production by PMNs. Bars show the mean ± SEM of PMNs from three donors. p values (two-way ANOVA, means compared to medium alone) are shown if p < 0.05.(I) Percentage of PMNs undergoing PMA-induced NETosis. The x axis indicates when NA-11 was added relative to the addition of PMA (t = 0). Bars show the mean ± SEM of PMNs from three donors. p values (one-way ANOVA, means compared to medium alone) are shown if p < 0.05.See also Figure S1.

Fig 5: Persistent hypoxia increases signaling molecules in pyroptosis pathway and enhances lactate dehydrogenase (LDH) in human primary trophoblasts.Primary trophoblast cells, isolated from the placentas from three deliveries with normal pregnancy at term, were treated with hypoxia (Hyp) or normoxia (Nor) for 3 days and lysed for immunoblotting. Hypoxia increases the expression of caspase-1, GSDMD, IL-18, IL-1ß, and NLRP3. Comparison of the signal intensity reveals that these molecules are significantly elevated in hypoxia-treated primary trophoblasts relative to control cells (n = 3, p < 0.05). c LDH colorimetric assay shows higher level of LDH in supernatant from hypoxia-treated cells vs. control cells (n = 6, p < 0.05). Data are presented as mean ± SD and analyzed by a Student’s t test.