Journal: Cell reports. Medicine

Article Title: NLRP3 inflammasome activation and symptom burden in KRAS-mutated CMML patients is reverted by IL-1 blocking therapy.

doi: 10.1016/j.xcrm.2023.101329

Figure Lengend Snippet: Figure 3. Monocytes from CMML KRASmut patients present a constitutive inflammasome activation (A) Percentage of ASC-specking monocytes in healthy controls (white bars), CMML patients without KRAS mutation (KRASwt, blue bars), and CMML patients with a KRAS mutation (KRASmut, orange bars) at baseline or treated as indicated for NLRP3 (LPS+ATP) or pyrin (LPS+TcdB) inflammasome activation. (B and C) Release of IL-1b from peripheral blood mononuclear cells (PBMCs) (B) and the formation of ASC specks in monocytes (C) in CMML KRASmut patients at baseline or after the indicated stimulation or treatment (LPS+ATP and LPS+TcdB for NLRP3 or pyrin inflammasome, respectively), in the absence/presence of MCC950. (B) Fold increase was calculated to control non-stimulated conditions, where the average of the higher value used to calculate fold increase is 1,066.58 pg/mL. (D) Percentage of extracellular LDH from untreated PBMCs from healthy donors (white bar), KRASwt patients (blue bar), and KRASmut patients (orange bar). Data are normalized to the percentage of monocytes. (E) Plasma concentration of HMGB1 and P2X7 receptor from healthy donors (white bar), KRASwt patients (blue bar), and KRASmut patients (orange bar). For (A)–(E), data are represented as mean ± SEM; each dot represents an individual patient; ordinary one-way ANOVA test (two-tailed) was used for (A) (* compares CMML KRASmut vs. healthy controls; y compares CMML KRASmut vs. CMML KRASwt), and two-tailed t test in (B)–(E). Note that * or yp < 0.05; **p < 0.01; ***p < 0.001; **** or yyyyp < 0.0001; ns, no significant difference (p > 0.05).

Article Snippet: Plasma or cell-free supernatants from fresh EDTA-anticoagulated PB samples from CMML patients and controls were used to quantify the concentration of human IL-1b (#BMS224INST, Invitrogen), human IL-18 (#7620, MBL), human IL-18BP (#EHIL18BP, Invitrogen), human TNF-a (#DTA00D, R&D Systems), human IL-6 (#D6050, R&D Systems), human ASC (#CSB-EL019114HU, Cusabio), human soluble P2X7 receptor (#CSB-EL017325HU, Cusabio), and human HMGB1 (#ARG81185, Arigo Biolaboratories) by ELISA following the manufacturer’s instructions.

Techniques: Activation Assay, Mutagenesis, Control, Clinical Proteomics, Concentration Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Macrophage Inflammasome Activation Drives Anti-inflammatory Responses via Neutrophil-Derived ASC Speck Transfer and Gasdermin-Dependent Caspase-1 Inhibition

doi: 10.1101/2025.01.22.634381

Figure Lengend Snippet: hASC and BSA (as control) were injected in the pericardium of wild-type (C, F), Tg(mpx:eGFP) (A, D) and Tg(mfap4:tomato) (B, E) larvae. In some experiments larvae were treated by immersion with DMSO/water, as controls, or with a specific inhibitor of aspase-1 (VX-765, 100 µM) (C) or 1 mg/ml colchicine (D, E, F). The transcript levels of the indicated genes (C, F) were analyzed at 24 hpi by RT-qPCR. Neutrophil (A, D) and macrophage (B, E) number and their recruitment were analyzed at 1, 3, 6 and 24 hpi by fluorescence microscopy. Each dot represents one individual, and the means ± SEM for each group is also shown. P values were calculated using one-way analysis of variance (ANOVA) and Tukey multiple range test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001. auf, arbitrary units of fluorescence.

Article Snippet: Recombinant hASC (#CSB-EP890936HU, Cusabio) was resuspended in sterile water at the concentration of 1 mg/ml.

Techniques: Control, Injection, Quantitative RT-PCR, Fluorescence, Microscopy

Journal: bioRxiv

Article Title: Macrophage Inflammasome Activation Drives Anti-inflammatory Responses via Neutrophil-Derived ASC Speck Transfer and Gasdermin-Dependent Caspase-1 Inhibition

doi: 10.1101/2025.01.22.634381

Figure Lengend Snippet: Two-dpf Tg(mpx:eGFP) (A) and Tg(mfap4:tomato) (B) larvae were injected in the pericardium with either hASC or BSA (control), then treated by immersion with 1 mg/ml colchicine. Neutrophil (A) and macrophage (B) number was analyzed by fluorescence microscopy at 1 and 3 hpi. The regions of interest used for quantification in all experiments are indicated in the schematic representation of the larvae. Bars: 100 µm (whole larva) and 50 µm (head).

Article Snippet: Recombinant hASC (#CSB-EP890936HU, Cusabio) was resuspended in sterile water at the concentration of 1 mg/ml.

Techniques: Injection, Control, Fluorescence, Microscopy

Journal: bioRxiv

Article Title: Macrophage Inflammasome Activation Drives Anti-inflammatory Responses via Neutrophil-Derived ASC Speck Transfer and Gasdermin-Dependent Caspase-1 Inhibition

doi: 10.1101/2025.01.22.634381

Figure Lengend Snippet: Tg (mpx:DN-caspa) ( A, B ), Tg (mpeg1:DN-caspa) ( C, D ) embryos were used. Wild type ( E, F, G H ) one-cell stage embryos were injected with gsdme and/or nlrp3 g RNAs/Cas9 complexes ( E, F, G ). At 2 dpf hASC was injected in the pericardium ( A, B, C, D ). At 2 dpf larvae were injected with ST in the yolk sac ( E, F, G ). The transcript levels of the indicated genes ( A-F ) were analyzed at 3 or 24 hpi by RT-qPCR. Caspase-1 activity was determined at 24 hpi using a fluorogenic substrate ( G ). Each dot represents one individual, and the means ± SEM for each group is also shown. P values were calculated using one-way analysis of variance (ANOVA) and Tukey multiple range test. ** P ≤ 0.01, *** P ≤ 0.001, and ****P ≤ 0.0001.

Article Snippet: Recombinant hASC (#CSB-EP890936HU, Cusabio) was resuspended in sterile water at the concentration of 1 mg/ml.

Techniques: Injection, Quantitative RT-PCR, Activity Assay