Fig 2: AIM2 inflammasome-driven emphysema in the gp130F/F and CS models is associated with augmented apoptosis of alveolar type II epithelial cells. (A) Representative photomicrographs of cross-sections of lungs from 6mo WT, gp130F/F (F/F), gp130F/F:Aim2-/- (F/F:Aim2), gp130F/F:Asc-/- (F/F:Asc), and gp130F/F:Il1r-/- (F/F:Il1r) mice subjected to TUNEL assay. Representative TUNEL-positive apoptotic lung cells are circled. (Scale bars, 25 µm.) (B and C) Graphs depict quantification of numbers of apoptotic (B) TUNEL-positive cells and (C) dual-labeled cleaved Caspase-3/SPC–positive alveolar type II epithelial cells per high-power field (HPF) in lungs of indicated 6mo mice. n = 6 per genotype. ***P < 0.001, ****P < 0.0001; one-way ANOVA. (D) qPCR (normalized for 18S rRNA) on lung tissue from WT mice exposed to acute CS (+CS) or sham air (-CS). n = 5 per group. *P < 0.05, **P < 0.01; Student’s t test. (E) Immunoblots on lung lysates from indicated groups from A for pro (31 kDa) and mature (17 kDa) IL-1ß. (F) Densitometric quantification of immunoblots from E for pro and mature IL-1ß, normalized against tubulin. n = 3 per group. **P < 0.01; Student’s t test. (G) IL-1ß ELISA on lung tissue lysates from indicated groups. n = 5 per group. *P < 0.05; Student’s t test. (H and J) Representative photomicrographs of TUNEL-stained cross-sections of lungs from (H) WT mice +CS or -CS, the former also treated with Sup ODN (15 mg/kg), or Aim2-/- mice (Aim2) +CS and (J) WT and Nlrp3-/- mice (Nlrp3) +CS, for 4 d. Representative TUNEL-positive apoptotic lung cells are circled. (Scale bars, 25 µm.) (I and K) Quantification of apoptotic TUNEL-positive cell numbers in lungs from mouse groups in (I) H and (K) J. n = 5 per group. *P < 0.05, **P < 0.01; one-way ANOVA.

Fig 3: Augmented expression and activation of AIM2 inflammasome components in emphysema patient lung tissues and sera. (A and B) qPCR on lungs of emphysema patients (EMPH, n = 15) or emphysema-free control individuals (CTL, n = 5). Expression data normalized for 18S rRNA. *P < 0.05; Student’s t test. (C) Correlation analyses of AIM2 and PYCARD gene expression in lungs of emphysema patients (n = 20). r, Pearson correlation coefficient. (D and F) Graphs depicting quantification of (D) AIM2- and (F) cleaved Caspase-1–positive staining cell numbers per high-power field (HPF) in lungs of emphysema patients (n = 10) or emphysema-free control individuals (n = 5), plus individuals stratified as nonsmokers (NS; n = 5) or current smokers (S; n = 5). *P < 0.05, **P < 0.01; Student’s t test. (E and G) Representative photomicrographs of lung cross-sections from emphysema patients or emphysema-free controls stained for human (E) AIM2 or (G) cleaved Caspase-1. Arrows depict representative positively stained cells. (Scale bars, 20 µm.) (H) Correlation analyses of immunohistochemical staining between AIM2 and cleaved Caspase-1 in lung sections from emphysema patients and emphysema-free controls (n = 14). r, Pearson correlation coefficient. (I) qPCR (normalized for 18S rRNA) on lungs of emphysema patients or emphysema-free controls from A and B. *P < 0.05; Student’s t test. (J and K) ELISA for (J) total IL-1ß and (K) mature IL-18 in emphysema patients (n = 12) or emphysema-free controls (n = 5). *P < 0.05; Mann–Whitney U test.

Fig 4: Involvement of the inflammasome in smoking mice.AIM2 expression was not altered in the lung of smoking and room air mice at 4 (A), 8 (B) and 16 weeks (C). (D) Quantitative analysis of western blot results was performed by means of ImageJ software (NIH, USA). Instead, AIM2 expression was higher in recruited DCs (identified as CD11c+CD11bintF4/80– cells) at 4 weeks (E) and macrophages (identified as CD11c+CD11bhighF4/80+ cells) at 16 weeks (F) post smoking exposure. (G) Caspase-1 was not active (25-10 kDa) in the groups of mice. (H) Quantitative analysis of the precursor form of caspase-1 compared to the loading control. (I, J, K) Caspase-11 was in its active form especially at 16 weeks in smoking mice compared to Room Air group, as shown in the quantitative analyses at Figure (L) (precursor form, p55 kDa) and (M) (active form, p25 kDa). Figure D, E, F, H, L, M show data as mean ± SEM and statistical analysis has been performed according to Two Way Anova followed by Sidak’s multiple comparison post-test. Experiments were repeated twice.

Fig 5: Increased mRNA and protein expression of inflammasome components in thyroid tissues from AIT patients. (A) The mRNA expression levels of NLRP1, NLRP3, NLRC4, absent in melanoma 2 (AIM2), ASC, and caspase-1 in thyroid tissues from AIT patients and controls (N = 50 in each group). Representative stripe images (B) and corresponding quantitative analysis (C) of the NLRP1, NLRP3, NLRC4, AIM2, ASC, caspase-1 and caspase-1 p20 protein levels in thyroid tissues from AIT patients and controls (N = 20 in each group). The relative mRNA and protein expression levels were corrected to GAPDH expression. The columns represent the mean ± SD values. Student’s t-test or Mann–Whitney U-test was used for comparison between the AIT and control groups. *P < 0.05, **P < 0.01. AIT, autoimmune thyroiditis; CON, controls.