Fig 2: The V-ATPase regulates the NLRP3 inflammasome in primary CD14+ monocytes. (A) Western blots of DSS cross-linked Nonidet P-40 (NP-40) insoluble ASC oligomers and NP-40 soluble ASC, pro-caspase-1 (p45), mature caspase-1 (p10), and lipidated LC3B, detected from human CD14+ monocyte total cell lysates stimulated with LPS (1 µg·mL-1) plus or minus bafilomycin A1 (BafA, 100 nm) for 18 h (n = 6). (B) Western blots of mature caspase-1 (p20) and LC3B detected from CD14+ monocyte total lysates (cell lysates + supernatants) stimulated with BafA (100 nm) or concanamycin A (ConA, 100 nm) plus or minus LPS (1 µg·mL-1) for 18 h (n = 4). (C) Representative immunofluorescence images of CD14 + monocytes immunostained for ASC in AlexaFluor 594 (red), with a DAPI (blue) nuclear co-stain after stimulation with LPS (1 µg·mL-1) plus and minus BafA (100 nm) for 18 h (scale bar is 25 µm, arrow heads denote ASC specks) (n = 4). (D) Western blot of mature caspase-1 (p20), pro-IL-1ß (p31), mature IL-1ß (p17) and ß-actin, from human CD14+ monocyte total cell lysate stimulated with LPS (1 µg·mL-1) in the presence of V-ATPase inhibitor bafilomycin A1 (BafA, 100 nm) or chymotrypsin inhibitor epoxomicin (Epo, 100 nm) (n = 3). (E) IL-1ß release and (F) cell death measured by ELISA and LDH assay respectively from the supernatants of CD14+ monocytes stimulated with LPS (1 µg·mL-1) and/or BafA (100 nm) for 18 h (n = 9). (G) IL-6, (H) TNF and (I) IL-1a release measured by ELISA, from human CD14+ monocyte supernatants stimulated with LPS (1 µg·mL-1) in the presence or absence of BafA, (100 nm) for 18 h (n = 9). *P < 0.05, ***P < 0.001; ns, not significantly different determined by a one-way ANOVA with Sidak’s post hoc comparison. Values shown are the mean ± SEM.

Fig 3: Inhibition of CASP1 attenuates ß-GP-induced VSMC pyroptosis and calcification.VSMCs were incubated in culture medium containing ß-GP (10 mM) for 7 days with or without CASP1 inhibitor (VX-765; 10 µM). A Protein levels of pro-CASP1, cleaved CASP1, GSDMD-N, and IL1B were determined by western blotting. B Quantification of the results shown in A. C The IL1B content in VSMC culture supernatants was determined by ELISA. D The release of LDH was detected using the LDH Assay Kit. E The percentage of PI-positive cells was measured using Hoechst 33342 (blue)/PI (red) double staining (top: Representative images; bottom: Quantitative analysis of PI-positive cells). Scale bar = 50 µm. F Calcium deposition in VSMCs was assessed by Alizarin red staining (positive staining: red; scale bar = 100 µm). G Quantitative analysis of calcium deposition in VSMCs normalized to the protein content. Mouse aortic rings were incubated in the culture medium containing ß-GP (10 mM) for 7 days with or without VX-765(10 µM). Calcification was assessed by Alizarin red staining (H), and calcium content of aortas was measured as described in methods (I). (positive staining: red; scale bar = 100 µm). Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 vs. control group; #P < 0.05, ##P < 0.01 vs. indicated group; and N.S. not significant.

Fig 4: A graphic illustration of the protective role of Irisin in CKD-associated VC.High phosphate (Pi) induces the production of ROS, which activates NLRP3 inflammasome, leads to the activation of CASP1-dependent pyroptotic cell death, and release of inflammatory factors in VSMCs, and ultimately promotes the formation of VC in CKD (Red arrows). Irisin induces autophagy and reduces ROS production. These effects lead to inhibition of NLRP3-mediated pyroptotic cell death, proinflammatory response, and calcium deposition in VSMCs, and ameliorate the progression of VC in CKD (Blue arrows).

Fig 5: - Adenosine synthase A restrains Th17 response via NLRP3 inflammasome and A2aR pathway. (a) Schematic diagram for S. aureus intraperitoneal reinfection model. (b) Survival of 3x reinfected BALB/c mice rechallenged with wild type (WT) USA300 strain [4 × 107 CFU, i.v. (intravenously)] for 14 days (n = 10 mice per group; *P < 0.05, Kaplan-Meier survival curves were generated, Log rank test was performed). (c) ELISA analysis of IL-17A and IFN-? in culture supernatant from splenocytes (harvested from 3x infection mice) restimulated by heat-killed S. aureus USA300 (MOI=5, 4 days) (n = 6 mice per group; ??P < 0.005, Mann-Whitney U test, Data are shown as mean ± SD). (d) ELISA analysis of IL-17A and IFN-? in culture supernatant from splenocytes (harvested from 1x infection mice) restimulated by heat-killed S. aureus USA300 (MOI=5, 4 days) (n = 6 mice per group; ??P < 0.005, Mann-Whitney U test, Data are shown as mean ± SD). (e) ELISA analysis of mean total and S. aureus-specific IgG levels in the serum from mice reinfected with WT strain or adsA mutant strain (4 × 107 CFU, i.p.) at day 17 (n = 8 mice per group; ??P < 0.005, ???P < 0.001, Mann-Whitney U test, Data are shown as mean ± SD). (f) CFU analysis of staphylococcal burden in kidneys from 3x reinfected BALB/c mice rechallenged with WT strain for 3 days (2 × 107 CFU, iv) (n = 6 mice per group; ??P < 0.005, Mann-Whitney U test, Data are shown as mean ± SD). (g) Representative kidney H&E-stained histologic sections at 3 days in the reinfection model, Scale bars, 100 µm. (h) ELISA analysis of IL-17A in culture supernatant from splenocytes (harvested from 3x infection mice treated with indicated inhibitors and vehicle control) restimulated by heat-killed S. aureus USA300 (MOI=5, 4 days) (n = 5 mice per group; *P<0.05, ??P < 0.005, ???P<0.001, ANOVA followed by Bonferroni correction, Data are shown as mean ± SD). (i) ELISA analysis of IL-17A in culture supernatant from splenocytes (harvested from 3x infection wild type C57BL/6 mice, Casp1-/- and Nlrp3-/- mice) restimulated by heat-killed S. aureus USA300 (MOI=5, 4 days) (n = 5 mice per group; ??P < 0.005, ???P<0.001, ANOVA followed by Bonferroni correction, Data are shown as mean ± SD). Data are representative of two independent experiments.