Fig 2: NSPs but not caspase-1 play a major role in pro-IL-1ß processing. (A upper part and B) Neutrophils were pre-treated for 30 min in the presence or absence of AEBSF (1 mM), Ac-YVAD-CMK (50 µM), VX-765 (50 µM) or their combination of AEBSF and one of the caspase-1 inhibitors, then cultured with or without LPS for 2 h and after were stimulated or not with ATP (2.5 mM) for another 1.5 h. Then, whole cell extracts were subjected to Western blot (A, B) and supernatants collected for ELISA quantification (A; bottom part). Western blots in A and B belong to the same cells extracts that were electrophoresed and electro-transferred twice, one probed with an anti-human IL-1ß antibody from Santa Cruz (antibody H-153; A) and the other one with a Novus Biological antibody (NB600-633; B). (A, upper part and B) Representative Western blot assay (n=6; A and n=2; B) of pro-IL-1ß and mature IL-1ß expression; and (A, bottom part) IL-1ß concentration in culture supernatants of the same cells from which lysates were immunoblotted in (A) and (B). (C, D) Concentrations determined by specific ELISAs of pro-IL-1ß (C) and IL-1ß (D) in culture supernatants and cell pellets of neutrophils stimulated in the absence or presence of AEBSF (0.35 mM), Ac-YVAD-CMK (50 µM), the combination of AEBSF and Ac-YVAD-CMK, or Z-VAD-FMK (50 µM) added 30 min before LPS stimulation, then stimulated or not for 2 h with LPS and then treated or not with ATP for another 1.5 h. Data represent the mean ± SEM of experiments with 3 (C) or 5 (D) donors performed in duplicate. *p<0.05 vs LPS+ATP; #p<0.05 vs Basal. One-way ANOVA with Sidak´s multiple comparisons test.

Fig 3: D + Q treatment prevents age-dependent SASP progression and promotes NP cell phenotype maintenance and disc cell viability.a–c”’ Immunohistological staining and staining area quantifications of SASP markers IL-1, IL-6, and MMP13 in the disc compartments of 1-year-old healthy Ctr (1 y Ctr), 14–23 M Veh-, and 14–23 M D + Q-treated mice. d–e”’ Quantitative immunostaining of NP cell phenotypic markers CA3 and GLUT1. f–f”’ Cell death assessment in the disc compartments by TUNEL staining. Two-tailed ANOVA or Kruskal–Wallis tests were used as appropriate; n = 6 mice/group, 2–3 levels per mouse. Data are represented as mean ± SEM. Scale bar = 200 µm. Source data are provided as a Source Data file.

Fig 4: Inflammasome-related proteins are present in extracellular vesicles isolated from human plasma samples.a Extracellular vesicles (EVs) were isolated from fresh plasma samples using the ExoQuick Plasma Prep with Thrombin (EXOQ5TM-1, System Biosciences, Palo Alto, CA). The EV fractions were lysed and sonicated before analysis. The soluble and EV fractions from 8 plasma samples were analyzed via SDS–PAGE and subsequent immunoblotting. Anti-NLRP3, anti-ASC, anti-CASP1, anti-GSDMD, and anti-IL-1B antibodies were used for the detection of inflammasome-related proteins. Anti-CD81 and anti-L1CAM antibodies were used to confirm the purity of the EV fraction and assess the presence of neural-derived EVs, respectively. b The presence of inflammasome-related proteins was most often found solely in the EV fraction and the detection of EV-specific markers was found only in the EV fraction. All statistical analyses were performed using unpaired Students t-tests (NLRP3, ****P < 0.0001) (ASC, ***P = 0.0002) (Pro-CASP1, ****P < 0.0001) (p20 CASP1, ****P < 0.0001) (Pro-GSDMD, ****P < 0.0001) (Cleaved GSDMD, ****P < 0.0001) (Pro IL-1B, ****P < 0.0001) (CD81, ****P < 0.0001) (L1CAM, **P = 0.0018). Error bars represent s.e.m.