Fig 2: NLRP3 (NOD-like receptor protein 3) inflammasome pathway was inhibited by colchicine treatment. A and B, Representative immunoblots and the corresponding analysis of NLRP3 inflammasome pathway in cardiac tissues at the end point of experiment. GAPDH shows as loading control. C, Representative immunofluorescence images of cardiac tissues staining with ASC (green), cTNT (cardiac troponin T) (red), and DAPI (blue). Frequency of ASC speck is shown on the right (n=6–8 each group). Five images per mouse are evaluated. Scale bar=50 µm. Data are shown as mean±SEM. All data statistical significance are determined by 1-way ANOVA (Tukey post-test). cTNT indicates cardiac troponin T; DCM, dilated cardiomyopathy; IL-1ß, interleukin-1ß; and NLRP3, NOD-like receptor protein 3. *P<0.05 between groups; **P<0.01 between groups; ***P<0.005 between groups; and ****P<0.001 between groups. AIM2 indicates absent in melanoma 2; ASC, apoptosis-associated speck-like protein with CARD domain; Col, colchicine.

Fig 3: Colchicine reduced the recruitment of neutrophils. A, ELISA analysis of inflammatory factor interleukin-1, interleukin-6, and tumor necrosis factor-a in murine plasma at the end point of experiment (n=8–12 each group). B, ELISA analysis of GDF15 in murine plasma at the end point of experiment (n=8–12 each group). C, Relative expression of inflammatory cytokine interleukin-1 mRNA, interleukin-6 mRNA, and tumor necrosis factor-a mRNA are normalized to GAPDH mRNA in murine cardiac tissues at the end point of experiment (n=5 each group). D and E, Representative flow cytometric and statistical analysis of CD11b+Ly6G+ neutrophil in the blood (D) and CD45+Ly6G+ neutrophil in the heart € (n=6 each group). F, Representative immunofluorescence images of cardiac tissues staining with Ly6G (green), cTNT (cardiac troponin T) (red), and DAPI (blue). Frequency of Ly6G-positive neutrophils is shown on the right (n=6–8 each group). Five images per mouse are evaluated. Scale bar=50 µm. Data are shown as mean±SEM. All data statistical significance are determined by 1-way ANOVA (Tukey post-test). cTNT indicates cardiac troponin T; DCM, dilated cardiomyopathy; GDF15, growth and differentiation factor 15; IL-1, interleukin-1; IL-6, interleukin-6; and TNF-a, tumor necrosis factor-a. *P<0.05 between groups; **P<0.01 between groups; ***P<0.005 between groups; and ****P<0.001 between groups. Col indicates colchicine; Ly6G, lymphocyte antigen 6 complex locus G6D.

Fig 4: Genetic inhibition of RTP801 levels in the dorsal hippocampus reduces the levels of inflammasome components NLRP1 and NLRP3 and procaspase 1.A, C Immunoblottings for NLRP1, cleaved NLRP1, and GFP as loading control for transduced neurons in the dorsal hippocampus of 7.5-month-old WT shCt, WT shRTP801, 5xFAD shCt, and 5xFAD shRTP801 groups of mice. B, D Densitometric quantification of NRLP1 and cleaved NLRP1 results as in (A, C) for the hippocampus (NLRP1 treatment effect: F(1, 46) = 8.714, P = 0.0050, interaction: F(1, 46) = 1.932, P = 0.1713; cleaved NLRP1 treatment effect: F(1, 46) = 15.84, P = 0.0003, interaction: F(1, 46) = 5.702, P = 0.0214). E, G Immunoblottings for NLRP3 and procaspase 1 and actin as loading control in the dorsal hippocampus of 7.5-month-old WT shCt, WT shRTP801, 5xFAD shCt, and 5xFAD shRTP801 groups of mice. F, H Densitometric quantification of NRLP3 and procaspase 1 results as in (E, G) for the hippocampus (NLRP3 treatment effect: F(1, 50) = 10.28, P = 0.0023, genotype effect: F(1, 50) = 14.72, P = 0.0004; procaspase 1 treatment effect: F(1, 51) = 8.438, P = 0.0054, genotype effect: F(1, 51) = 17.29, P = 0.0001). Data are means ± SEM. In all panels two-way ANOVA with Bonferroni’s post hoc test was performed: *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 5: Colchicine promotes the deacetylation of NLRP3 (NOD-like receptor protein 3) by SIRT2 (Sirtuin 2). A, Representative immunofluorescence images of cardiac tissues staining with NLRP3 (green), SIRT2 (red), and DAPI (blue). (n=6–8 each group). Five images per mouse are evaluated. Scale bar=50 µm. B, Representative immunofluorescence images of primary neutrophils that are treated +/- colchicine (10 µmol/L, 2 hours), +/- SIRT2 inhibitor AGK2 (10 µmol/L, 2 hours) or challenged with doxorubicin (5 µmol/L, 2 hours) and inflammasome activators nigericin (10 µmol/L, 2 hours). NLRP3 (green), SIRT2 (red) and DAPI (blue). Scale bar=20 µm. Data are representative of 3 independent experiments. C, The effect of SIRT2 on the deacetylation of NLRP3 is evaluated by coimmunoprecipitation. NLRP3-Flag plasmid is transfected into NB4 cell line, then NB4 cell line is treated with +/- colchicine (10 µmol/L, 2 hours), +/- SIRT2 inhibitor AGK2 (10 µmol/L, 2 hours) or challenged with doxorubicin (5 µmol/L, 2 hours) and inflammasome activators nigericin (10 µmol/L, 2 hours). Cell lysates are immunopurified with anti-Flag antibody. Then the expression of acetylated lysine is measured by IP. GAPDH is the input loading control. Data are representative of 3 independent experiments. DCM indicates dilated cardiomyopathy; IL-1ß, interleukin-1ß; NLRP3, NOD-like receptor protein 3; and SIRT2, Sirtuin 2. AGK2, a reversible inhibitor of SIRT2; Col, colchicine; IB, immunoblot; IP, Immunoprecipitation.