Fig 2: F4/80+ cells in CNV lesions have high levels of caspase-1 protein.(A) Immunolabeling of eye sections of Vegfahyper mice with an antibody that recognizes endogenous levels of caspase-1 protein only when cleaved at Asp296 that occurs with inflammasome activation but does not detect full-length caspase-1 (Cell Signaling Technology Cat# 89332). F4/80+ cells at the site of a CNV lesion (white) show the presence of cleaved caspase-1 (green, white arrow), whereas this staining is not observed in RPE cells (red arrow) in eyes of Vegfahyper mice. DAPI nuclear staining in blue. Scale bar, 50 μm. Location of the outer nuclear layer of the retina (ONL), choroid, and RPE are shown. (B) Immunolabeling of choroidal flat mounts for caspase-1 p10 (green) shows predominant staining in F4/80+ cells (white, arrowheads) but not in RPE cells (arrows) in CNV lesions. The antibody (sc-22166, Santa Cruz Biotechnology) detects cleaved caspase-1 p10 (inflammasome activation marker) and was raised against a short amino acid sequence containing the neoepitope at Gly315 of caspase-1 of mouse origin. Proteolytic cleavage of the precursor caspase-1 at Gly315 generates the functional caspase-1 subunits, known as p20 and p10 subunits. Choroidal flat mounts from 3-months-old Vegfahyper, VegfahyperNlrp3-/-, and VegfahyperBest1Cre/+Nlrp3A350V/A350V mice are shown. No caspase-1 staining is observed even in aged VegfahyperCasp1-/-Casp11-/- mice (red arrowhead), confirming the specificity of the caspase-1 immunolabeling (a representative flat mount of an 18-months-old mouse is shown). DAPI nuclear staining in blue. Right: Higher magnification images of F4/80+ cells (highlighted in boxes of left images). Scale bars, 20 μm (left) and 10 μm (right). (C) At sites of CNV lesions in mice with the Vegfahyper allele, a subset of infiltrating F4/80+ cells shows immunolabeling for caspase-1 p10 and is also positive for the microglial marker P2RY12 (green arrows). In contrast, other F4/80+p10+ cells show no staining for P2RY12 (yellow arrows). Representative image of a 12-months-old mouse eye with the Vegfahyper allele is shown (here an eye of a VegfahyperC3-/- mouse is shown). Scale bars, 50 μm. DAPI nuclear staining in blue. Location of ONL, choroid, and RPE are shown.

Fig 3: Inflammasome signaling plays important roles in cancer cells and TAM interaction. (A) Immunohistochemistry and quantitative correlation analyses of AIM2 and NLRP3 expression and IBA-1+ TAM infiltration in GBM patients. Scale bar: 25 μm. (B and C) Western blotting analysis of the inflammasome (NLRP3 and AIM2), C-Caspase 1 (cleaved caspase 1), and PD-L1 expression in THP-1 co-cultured GBM and BRCA cells, pre-treated with MB (B) or CASP1 targeted siRNA (C). (D and E) Migration analysis of THP-1, co-cultured with cancer cells that were pre-treated with MB (D) or CASP1 targeted siRNA (E). Scale bar: 25 μm. (F and G) Western blotting analysis of PD-L1 expression in THP-1 which was co-cultured with cancer cells pre-treated with MB (F) or CASP1 targeted siRNAs (G). **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig 4: C. albicans-induced GSDMD cleavage and C. albicans escape from macrophages relies on inflammasome activation.a Casp1/11-mediated C. albicans-induced BMDM death. BMDMs from WT and Casp-1/11-/- mice were infected with C. albicans at MOI 10. BMDM death at the indicated time points was measured as relative LDH release as described in Fig. 3f. Two-tailed unpaired Student’s t test was used for statistical comparisons. Data shown are means ± SD (n = 3 biologically independent samples per group). p = 0.0016 (4 h), p = 0.0282 (8 h), p = 0.0172 (16 h) vs. WT. b Casp1/11-mediated C. albicans escape from macrophages. BMDMs from WT and Casp-1/11-/- mice were infected with C. albicans at MOI 10. Dying cells were stained with PI (red). Experiments were repeated three times. The related time-lapse video is included in the Supplemental Materials. c The percentage of PI-positive BMDMs calculated at each time point. At least 100 cells were assessed for each sample. Two-tailed unpaired Student’s t test was used for statistical comparisons. Data shown are means ± SD (n = 3 biologically independent samples per group). p = 0.0484 (3 h), p = 0.0257 (6 h) vs. WT. d Potassium efflux was essential for C. albicans-triggered GSDMD cleavage in BMDMs. BMDMs from WT or Gsdmd-/- mice were cultured in the indicated concentrations of KCl for 1 h and then infected with WT C. albicans for 6 h at MOI 50. Full-length (p53) and cleaved (p30) GSDMD in the cell lysates were detected by western blotting. Results are representative of data from three biological replicates. Source data are provided as a Source Data file. e C. albicans-induced BMDM death assessed by LDH assay. BMDMs were cultured in the indicated concentrations of KCl for 1 h and then infected (uninfected BMDMs as controls) with WT, candidalysin-deficient (ece1Δ/Δ), or hyphae-deficient (cph1Δ/Δ, efg1Δ/Δ) C. albicans at MOI 10 for 12 h. Relative LDH release was assessed as described in Fig. 3f. Two-tailed unpaired Student’s t test was used for statistical comparisons. Data shown are means ± SD (n = 3 biologically independent samples per group). For cph1Δ/Δ, efg1Δ/Δ-infected BMMCs, p = 0.0016 (KCl 50 mM) vs. KCl untreated macrophages. For KCl -untreated BMMCs, p < 0.0001 (ece1Δ/Δ, cph1Δ/Δ, efg1Δ/Δ, w/o C. albicans) vs. WT C. albicans. For KCl 30 mM -treated BMMCs, p = 0.0001 (cph1Δ/Δ, efg1Δ/Δ), p < 0.0001 (ece1Δ/Δ, w/o C. albicans) vs. WT C. albicans. For KCl 50 mM-treated BMMCs, p = 0.0001 (ece1Δ/Δ), p < 0.0001 (cph1Δ/Δ, efg1Δ/Δ, w/o C. albicans) vs. WT C. albicans. f Candidalysin-dependent LDH release calculated by subtracting the level of LDH release induced by candidalysin-deficient (ece1Δ/Δ) C. albicans from that induced by WT C. albicans. Two-tailed unpaired Student’s t test was used for statistical comparisons. Data shown are means ± SD (n = 3 biologically independent samples per group). p = 0.0052 (KCl 30 mM), p = 0.0036 (KCl 50 mM) vs. KCl untreated macrophages. g Potassium efflux was critical for C. albicans escape from macrophages. BMDMs were cultured in the indicated concentrations of KCl for 1 h and then infected with WT C. albicans at MOI 10. Dying cells were stained with PI (red). Images were acquired as described in Fig. 3c. Shown are representative images at the indicated time points. The related time-lapse video is included in the Supplemental Materials. h The percentage of PI-positive BMDMs calculated at each time point. At least 100 cells were assessed for each sample. Two-tailed unpaired Student’s t test was used for statistical comparisons. Data shown are means ± SD (n = 3 biologically independent samples per group). p = 0.016 (KCl 50 mM) vs. KCl untreated macrophages.

Fig 5: MPP+-induced inflammation of HMC3 microglia and anti-inflammatory potentials of NC009 compounds. (A) Cytotoxicity of MPP+ against HMC3 cells by MTT and NO release assays. Cells were treated with MPP+ (0–10 mM) and cell viability and NO release in culture medium was measured the following day (n = 3). To normalize, the relative cell viability in MPP+-untreated (inactive) cells was set at 100%. p values: comparisons between inactive and activated cells (* p < 0.05, *** p < 0.001). (B) Experimental flow chart. HMC3 cells were plated on day 1. After 20 h, cells were pre-treated with test compounds (1–10 μM) for 8 h, followed by MPP+ (3 mM) treatment for 20 h. On day 3, the HMC3 cells were examined for cell viability (MTT assay) and NO release (Griess assay) in culture medium. In addition, cells with 10 μM compound treatment were examined for CD68 and CD11B expression (ICC staining), IL-1β, IL-6, TNF-α release (ELISA), and NLRP3, CASP1, iNOS, IL-1β, IL-6, and TNF-α expression (Western). (C) Cell viability and NO release assays (n = 3). The relative cell viability in MPP+-untreated cells was normalized as 100%. (D) CD68 and CD11B expression (n = 3). Cells were analyzed by immunofluorescence using antibodies against CD68 (yellow) and CD11B (green). Cell nuclei were counterstained with DAPI (blue). (E) IL-1β, IL-6, and TNF-α release assays (n = 3). (F) NLRP3, CASP1, iNOS, CREB, IL-1β, IL-6, and TNF-α levels analyzed by immunoblotting using GAPDH as a loading control (n = 3). To normalize, protein expression level in MPP+-untreated cells was set at 100%. (C–F), p values: comparisons between MPP+-untreated and treated cells (# p < 0.05, ## p < 0.01, ### p < 0.001), or with and without compound addition (* p < 0.05, ** p < 0.01, *** p < 0.001).