Fig 2: Mutagenesis of D20 and V46 sites does not entirely block IL-37 Cleavage. (A) HEK293 cells were transfected with pIL-37b-myc/pIL-1ß-flag with/without pCaspase-1 and pASC. (Ai) Western blot analysis was carried out on cell lysates for Myc tag, Caspase-1, as well as loading control ß-actin. (Aii) Western blot analysis was carried out on cell lysates for flag tag, Caspase-1, as well as loading control ß-actin. (B) HEK293 cells were transfected with pIL-37b-Myc WT or V46G mutant with/without pCaspase-1 and pASC. Western blot analysis was carried out on cell lysates for Myc tag, Caspase-1, as well as loading control ß-actin. (C) mCherry alone (#mCherry) or IL-37b-mCherry WT, D20A or V46G THP-1 cells were treated with nigericin (10 µM, 45 min). Western blotting was carried out using an anti-IL-37 antibody (R&D). Data shown are representative of three independent experiments.

Fig 3: NLRP3 inflammasome activation mediates the release of IL-37. (A) Primary human monocytes were primed with LPS (1 µg/mL, 4 h), preincubated with MCC950 (1 µM) or Z-VAD (50 µM) for 15 min prior to treatment with nigericin (10 µM, 45 min). (Ai) Western blot analysis was carried out for mIL-37 (17.5 kDa), pro-IL-37 (27.5 kDa), mIL-1ß (17 kDa), pro-IL-1ß (31 kDa), as well as loading control ß-actin (42 kDa). (Aii) Levels of mIL-37 release were quantified using densitometry and standardized to mIL37 release after nigericin alone treatment. n = 3. **p < 0.01 using one-way ANOVA. (B) WT or NLRP3 KO THP-1 were primed with LPS (1 µg/mL, 4 h) and treated with nigericin (10 µM, 45 min). (Bi) Western blot analysis was carried out for mIL-37 (17.5 kDa), pro-IL-37 (27.5 kDa), mIL-1ß (17 kDa), pro-IL-1ß (31 kDa), NLRP3 (113 kDa), as well as loading control ß-actin (42 kDa). (Bii) Levels of mIL-37 release were quantified using densitometry and standardized to nigericin alone treatment. n = 3. **p < 0.01 using t-test. (C) IL-37b-mCherry expressing THP-1 cells were preincubated with MCC950 (1 µM) or Z-VAD (50 µM) for 15 min prior to treatment with nigericin (10 µM, 45 min). (D) Cherry alone (#mCherry) or IL-37b-mCherry THP-1 cells were stimulated with nigericin (10 µM, 45 min). The supernatant was concentrated, and RFP-trap immunoprecipitation was carried out. Input and immunoprecipitated fraction were ran on SDS-PAGE gel. Western blot analysis was carried out for IL-37b-mCherry using anti-IL-37 antibody (R&D). Blots are representative of at least three independent biological experiments. Data are shown as mean ± SD.

Fig 4: D20A and V46G mutants do not alter IL-37b localization. (A) THP-1 cells were stimulated with LPS (1 µg/mL, 18 h) or nigericin (10 µM, 45 min). Subcellular fractionation was performed. Western blot analysis for mIL-37 (17.5 kDa), pro-IL-37 (27.5 kDa), histone H3 (15 kDa), and GAPDH (36 kDa). All blots are representative of at least two independent experiments. (B) HEK293 or COS7 cells were transfected with pIL-37b-Myc with/without pCaspase-1 and pASC. Representative confocal images of Myc (red), ASC (green), and Dapi (blue) are shown. (C) Images from an immunofluorescence experiment showing WT/D20A/V46G IL-37b-mCherry overexpressing THP-1 cells stimulated with LPS (1 µg/mL, 18 h) or nigericin (10 µM, 30 min, unprimed). Representative confocal images of mCherry (red) and Dapi (blue) are shown. Images are representative of three independent biological experiments. Scale bar is 10 µm.

Fig 5: Long-term LPS treatment leads to IL-37 release dependent on membrane permeability. (A) Primary human monocytes or (B) THP-1s were pretreated with BFA (10 µg/mL) or punicalagin (50 µM) for 15 min prior to 18-h stimulation with LPS (1 µg/mL). (Ai) Secreted TNF-a and IL-6 were measured by ELISA. (Bi) Cell death was measured by LDH assay and shown as percentage relative to total cell death. n = 4–6. **p < 0.01; ***p <0.001 using one-way ANOVA. (Aii, Bii) Western blot analysis for mIL-37 (17.5 kDa), pro-IL-37 (27.5 kDa), mIL-1ß (17 kDa), pro-IL-1ß (31 kDa), as well as loading control ß-actin (42 kDa). (Aiii, Biii) Levels of mIL-37 release were quantified using densitometry and standardized to LPS treatment alone. n = 3. **p < 0.01; ***p < 0.001 using one-way ANOVA. (C) WT and GSDMD KO THP-1s were stimulated with LPS (1 µg/mL, 18 h). (Ci) Western blot analysis for mIL-37 (17.5 kDa), pro-IL-37 (27.5 kDa), mCaspase-1 (20 kDa), pro-Caspase-1 (45 kDa), GSDMD full length (FL, 53 kDa), GSDMD N-terminus (NT, 31 kDa), as well as loading control ß-actin (42 kDa). (Cii) mIL-37 release was quantified using densitometry and standardized to LPS treatment of WT cells. n = 3. *p < 0.05 using t-test. All blots are representative of at least three independent biological experiments. ns = not significant. Data are shown as mean ± SD.