Journal: Cell Reports Medicine

Article Title: Artificial cells delivering itaconic acid induce anti-inflammatory memory-like macrophages to reverse acute liver failure and prevent reinjury

doi: 10.1016/j.xcrm.2023.101132

Figure Lengend Snippet: AI-Cells controlled liver injury by inhibiting macrophage NLRP3 inflammasome activation and reducing IL-1β production (A) The levels of IL-1β, TNF-α, and IL-6 in the supernatant of BMDMs/Kupffer cells in various groups (n = 3 biological replicates). (B) Schematic representation of a BMDM/Kupffer cell inflammation model responding to TNF-α, IL-6, and IL-1β. AI-Cell treatment significantly reduced the concentration of IL-1β but had no significant effect on TNF-α and IL-6. (C) Quantification of viable AML-12 cells after IL-1β exposure (n = 5 biological replicates). The AML-12 cells were cultured in the B-AI-Cells CM supplemented with recombinant IL-1β. B-LPS&ATP CM was set up as a control. (D) Survival of AML-12 cells after K-LPS&ATP CM and IL-1β antibody exposure (n = 3 biological replicates). K-LPS&ATP CM was set up as a control. (E) Representative microscopy images of AML-12 cells treated with various conditioned medium from Kupffer cells and stained with crystal violet. Scale bars: 1,000 μm. (F) Western blot of NLRP3, pro-Casp1, Casp1 p20, and β-actin with protein lysates from liver tissues of sham, APAP, and AI cells groups. (G) Western blot of IL-1β with protein lysates from liver tissues of sham, APAP, and AI-Cells groups. (H–K) Densitometric analysis of NLRP3, pro-Casp1, Casp1 p20, and IL-1β proteins by ImageJ software (n = 3 biological replicates). (L) IL-1β staining of liver tissues from the sham, APAP, or AI-Cell-treated ALF mice after euthanasia. Scale bars: 200 μm. (M) Schematic diagram of AI-Cells blocking activation of NLPR3 inflammasome. (N) Docking mode of NLRP3 (white surface and green cartoon) with itaconic acid (cyan sticks). (O) Three-dimensional (3D) interaction mode of NLRP3 (green cartoon) and itaconic acid (cyan sticks). The key residues are shown as green sticks and the hydrogen bond is shown in yellow dashed lines. All data are expressed as mean ± SD; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, and ns is p > 0.05 by one-way ANOVA.

Article Snippet: Anti-pro-Casp1/anti-Casp1 p20 antibody (mouse) , Cell Signaling Technology , Cat# 89332; RRID: AB_2923067.

Techniques: Activation Assay, Concentration Assay, Cell Culture, Recombinant, Control, Microscopy, Staining, Western Blot, Software, Blocking Assay

Journal: Cell Reports Medicine

Article Title: Artificial cells delivering itaconic acid induce anti-inflammatory memory-like macrophages to reverse acute liver failure and prevent reinjury

doi: 10.1016/j.xcrm.2023.101132

Figure Lengend Snippet:

Article Snippet: Anti-pro-Casp1/anti-Casp1 p20 antibody (mouse) , Cell Signaling Technology , Cat# 89332; RRID: AB_2923067.

Techniques: Recombinant, RNA Extraction, Staining, Real-time Polymerase Chain Reaction, Software

Journal: Theranostics

Article Title: Therapeutic potential of garlic chive-derived vesicle-like nanoparticles in NLRP3 inflammasome-mediated inflammatory diseases

doi: 10.7150/thno.60265

Figure Lengend Snippet: GC-VLNs specifically inhibited NLRP3 inflammasome activation. ( A-D ) BMDMs were preincubated with GC-VLNs for 16 h, primed with LPS for 3 h, and stimulated with FFA sodium palmitate for 12 h to activate the NLRP3 inflammasome. Cells were lysed for immunoblot analysis of Casp1 p10 ( A ), and the cell-free media were subjected to enzyme-linked immunosorbent assay (ELISA) analysis to measure the release of IL-1β ( B ) and IL-18 ( C ). Pyroptotic cell death was assessed by measuring the level of lactate dehydrogenase (LDH) released in the culture media ( D ). ( E ) Immunoblot analysis of Casp1 p10 in lysates of peritoneal macrophages preincubated with GC-VLNs for 16 h, followed by NLRP3 inflammasome activation using LPS and FFA. ( F ) Immunoblot analysis of Casp1 p10 in cell lysates of BMDMs preincubated with GC-VLNs for 16 h, followed by AIM2 inflammasome activation. ( G ) Immunoblot analysis of Casp1 and IL-1β in cell lysates and culture media of BMDMs preincubated with GC-VLNs for 16 h, primed with LPS for 3 h, and stimulated with ATP for 30 min to activate the NLRP3 inflammasome. ( H ) Immunoblot analysis of Casp1 p10 in lysates of LPS-primed BMDMs, in which the NLRP3 inflammasome was activated by nigericin (Ni) or alum. 9×10 10 mL -1 of GC-VLNs were used to pretreat the cells for 16 h. Results were expressed as mean±SEM from three independent experiments. * (p < 0.05) and ** (p < 0.01) compared with LPS+FFA group (black bar). Tubulin was included to show equivalent loading in immunoblot analysis. LPS-treated macrophages were used as a negative control for inflammasome activation.

Article Snippet: Primary antibodies used were anti-NLRP3 mouse antibody (Adipogen, AG20B0014C100, 1:1000); anti-ASC rabbit antibody (Adipogen, AG25B0006C100, 1:1000); anti-Casp1 (p10) mouse antibody (Adipogen, AG20B0044C100, 1:1000); anti-tubulin rabbit polyantibody (Santa Cruz, Dallas, TX, USA, SC-5286, 1:200); anti-Nek7 rabbit antibody (Abcam, Cambridge, MA, USA, ab133514, 1:10000); and anti-IL-1β goat antibody (R&D systems, Minneapolis, MN, USA, AF401NA, 1:2000).

Techniques: Activation Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Negative Control

Journal: Theranostics

Article Title: Therapeutic potential of garlic chive-derived vesicle-like nanoparticles in NLRP3 inflammasome-mediated inflammatory diseases

doi: 10.7150/thno.60265

Figure Lengend Snippet: GC-VLNs alleviated inflammation in chemical-induced acute liver injury in mice. 8-week-old male C57BL/6J mice were intraperitoneally injected with the solvent phosphate-buffered saline (PBS) or GC-VLNs in PBS at 1×10 10 g -1 . 48 h later, acute liver injury was induced by intraperitoneal injection of GalN/LPS mixture. The mice were sacrificed 6 h after GalN/LPS injection. N = 4/group. ( A ) Representative images of liver sections with hematoxylin and eosin (H&E) staining. ( B ) Levels of AST and ALT in serum. ( C ) Levels of IL-1β in serum. ( D ) Levels of IL-18 in serum. ( E ) Relative mRNA levels of Nlrp3, Pycard, Casp1 , and Il1b genes in the livers. The housekeeping gene hypoxanthine guanine phosphoribosyl transferase ( Hprt ) was used to normalize mRNA levels. ( F ) Immunoblot analysis of liver lysates. Data were presented as mean±SEM. * (p < 0.05) and ** (p < 0.01) compared with the control group (bar with white squares).

Article Snippet: Primary antibodies used were anti-NLRP3 mouse antibody (Adipogen, AG20B0014C100, 1:1000); anti-ASC rabbit antibody (Adipogen, AG25B0006C100, 1:1000); anti-Casp1 (p10) mouse antibody (Adipogen, AG20B0044C100, 1:1000); anti-tubulin rabbit polyantibody (Santa Cruz, Dallas, TX, USA, SC-5286, 1:200); anti-Nek7 rabbit antibody (Abcam, Cambridge, MA, USA, ab133514, 1:10000); and anti-IL-1β goat antibody (R&D systems, Minneapolis, MN, USA, AF401NA, 1:2000).

Techniques: Injection, Solvent, Saline, Staining, Western Blot, Control

Journal: Theranostics

Article Title: Therapeutic potential of garlic chive-derived vesicle-like nanoparticles in NLRP3 inflammasome-mediated inflammatory diseases

doi: 10.7150/thno.60265

Figure Lengend Snippet: Lipids from active and inactive GC-VLNs were subjected to lipidomic analysis. ( A-C ) BMDMs were preincubated with liposomes prepared from lipids of active or inactive GC-VLNs, followed by NLRP3 inflammasome activation. ( A ) Immunoblot analysis of Casp1 p10 in cell lysates. ( B ) Levels of IL-1β in the culture media. ( C ) Levels of IL-18 in the culture media. Results were expressed as mean±SEM from three independent experiments. * (p < 0.05) and ** (p < 0.01) compared with LPS+ATP group (black bar). ( D ) Lipidome comparison of active and inactive GC-VLNs. PC: phosphatidylcholine; PE: phosphatidylethanolamine; PA: phosphatidic acid; PG: phosphatidylglycerol; MGDG: monogalactosyldiacylglycerol; DGDG: digalactosyldiacylglycerol. ( E ) Abundance and fold change of top eight PC species in active GC-VLNs.

Article Snippet: Primary antibodies used were anti-NLRP3 mouse antibody (Adipogen, AG20B0014C100, 1:1000); anti-ASC rabbit antibody (Adipogen, AG25B0006C100, 1:1000); anti-Casp1 (p10) mouse antibody (Adipogen, AG20B0044C100, 1:1000); anti-tubulin rabbit polyantibody (Santa Cruz, Dallas, TX, USA, SC-5286, 1:200); anti-Nek7 rabbit antibody (Abcam, Cambridge, MA, USA, ab133514, 1:10000); and anti-IL-1β goat antibody (R&D systems, Minneapolis, MN, USA, AF401NA, 1:2000).

Techniques: Liposomes, Activation Assay, Western Blot, Comparison

Journal: Theranostics

Article Title: Therapeutic potential of garlic chive-derived vesicle-like nanoparticles in NLRP3 inflammasome-mediated inflammatory diseases

doi: 10.7150/thno.60265

Figure Lengend Snippet: DLPC was identified to suppress NLRP3 inflammasome activation. ( A-B ) BMDMs were preincubated with liposomes prepared from PC lipids for 16 h, followed by LPS+ATP treatment to activate the NLRP3 inflammasome. ( A ) Immunoblot analysis of Casp1 p10 in cell lysates. ( B ) Levels of IL-1β in the culture media. ( C-H ) BMDMs were preincubated with DLPC liposomes (PC(36:4)) for 16 h, followed by LPS+ATP or LPS+FFA treatment to activate the NLRP3 inflammasome. Cell lysates were subjected to immunoblot analysis and cell-free media were used for cytokine measurement. Results were expressed as mean±SEM from three independent experiments. * (p < 0.05) and ** (p < 0.01) compared with LPS+ATP group or LPS+FFA group (black bar).

Article Snippet: Primary antibodies used were anti-NLRP3 mouse antibody (Adipogen, AG20B0014C100, 1:1000); anti-ASC rabbit antibody (Adipogen, AG25B0006C100, 1:1000); anti-Casp1 (p10) mouse antibody (Adipogen, AG20B0044C100, 1:1000); anti-tubulin rabbit polyantibody (Santa Cruz, Dallas, TX, USA, SC-5286, 1:200); anti-Nek7 rabbit antibody (Abcam, Cambridge, MA, USA, ab133514, 1:10000); and anti-IL-1β goat antibody (R&D systems, Minneapolis, MN, USA, AF401NA, 1:2000).

Techniques: Activation Assay, Liposomes, Western Blot