Journal: Cells

Article Title: Toxoplasma gondii Rhoptry Protein 7 (ROP7) Interacts with NLRP3 and Promotes Inflammasome Hyperactivation in THP-1-Derived Macrophages

doi: 10.3390/cells11101630

Figure Lengend Snippet: ROP7 in THP-1 cells activated NLRP3 inflammasome and induced a hyperactive status in cells. THP-1 Mock and THP-1 ROP7 were induced by 1-μg/mL doxycycline for 48 h and differentiated under 100-nM PMA for another 48 h. THP-1 infected with lentivirus as a mock control, THP-1 Mock . ( A ) NLRP3 and ASC were co-precipitated with Flag-HA-ROP7 in THP-1 cells. After expression and differentiation, the cells were harvested for Co-IP using an anti-HA antibody to immunoprecipitated Flag-HA-ROP7 and further analyzed by Western blot using an anti-NLRP3, anti-ASC, and anti-Flag antibody. ( B ) After differentiation, cell medium was renewed and collected periodically to detect IL-1β and TNF-α secretion by ELISA. Data are expressed as mean ± SEM values. * p < 0.05, *** p < 0.001, as compared with THP-1 Mock . ( C ) pro-IL-1β and p17 of THP-1 Mock and THP-1 ROP7 in cell lysate and supernatant (SN) were detected by immunoblotting using anti-IL-1β. In total, 100-ng/mL LPS was used in THP-1 Mock for 4 h as a positive control. ( D ) At 24 h post-induction, the cells were treated with 100-ng/mL LPS for 4 h alone or 10-μM nigericin for 1 h subsequently. Supernatants were collected for ELISA. Data are expressed as mean ± SEM values. ** p < 0.01, *** p < 0.001, as compared with THP-1 Mock . ( E ) After PMA differentiation, 10-μM of Z-YVAD-FMK, Z-VAD-FMK, or MCC950 was added to the cells for 24 h. The IL-1β and TNF-α in cell-free supernatant were detected by ELISA. Data are expressed as mean ± SEM values. *** p < 0.001, as compared with the DMSO group. ( F ) Supernatants that were untreated (24 h after PMA differentiation and medium replacement), LPS-treated (100 ng/mL for 4 h), and nigericin-treated (10 μM for 1 h) were collected for the detection of LDH release. The results were normalized by the maximum enzyme activity in each group. Data are expressed as mean ± SEM values. ns: non-significant, ** p < 0.01.

Article Snippet: Antibodies: anti-IL-1β (NB600-633, Novus Biologicals, Littleton, CO, USA); anti-HA (Ab137838, Abcam, Cambridge, UK); anti-Flag (F1804, Sigma-Aldrich, St Louis, MO, USA); anti-NLRP3 (AG-20B-0014-C100, Adipogen, San Diego, CA, USA); anti-ASC (AG-25B-0006-C100, Adipogen, San Diego, CA, USA) Reagents: recombinant human IL-1β (IL038, Sigma-Aldrich, St Louis, MO, USA), recombinant human TNF-α (ab9642, Abcam, Cambridge, UK); Z-YVAD-FMK (S8507, Selleck, Shanghai, China); Z-VAD-FMK (S7023, Selleck, Shanghai, China); MCC950 (S8930, Selleck, Shanghai, China); Bay 11-7082 (S2913, Selleck, Shanghai, China); IL-1R antagonist (sc-221747, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Infection, Control, Expressing, Co-Immunoprecipitation Assay, Immunoprecipitation, Western Blot, Enzyme-linked Immunosorbent Assay, Positive Control, Activity Assay

Journal: Cells

Article Title: Toxoplasma gondii Rhoptry Protein 7 (ROP7) Interacts with NLRP3 and Promotes Inflammasome Hyperactivation in THP-1-Derived Macrophages

doi: 10.3390/cells11101630

Figure Lengend Snippet: The NF-κB signaling pathway was activated in THP-1 ROP7 , but its activation was not directly derived from ROP7. THP-1 Mock and THP-1 ROP7 were induced by 1-μg/mL doxycycline for 48 h and differentiated under 100-nM PMA for another 48 h ( A ) or with 10-μM BAY11-7082 in THP-1 ROP7 . ( B ) Total RNA was extracted, and the mRNA relative fold changes were determined via RT-qPCR. ( C ) The 293T cells were co-transfected with a dual-luciferase reporter plasmid (ranilla: firefly 1:10) for NF-κB. pcDNA3-HA-ROP7 or empty plasmids (mock). At 48 h post-transfection, luciferase activity was examined, and the values were normalized according to the ratio of firefly luciferase activity and ranilla luciferase activity. In total, 20-ng/mL TNF-α was used as a positive control. ( D ) THP-1 ROP7 cells were induced by 1-μg/mL doxycycline for 48 h and differentiated under 100-nM PMA for another 48 h along with 10-μM Z-YVAD-FMK, Z-VAD-FMK, or MCC950. Total RNA was extracted, and the mRNA relative fold changes were determined via RT-qPCR. ( E ) THP-1 Mock and THP-1 ROP7 were induced by 1-μg/mL doxycycline for 48 h and differentiated under 100-nM PMA for another 48 h. Then, 100-ng/mL LPS for 4 h and 10-μM nigericin for 1 h subsequently were used to activate NLRP3 inflammasomes. In total, 10-μM Z-YVAD-FMK, Z-VAD-FMK, or MCC950 was added with LPS or nigericin. The IL-1β and TNF-α supernatant concentrations were detected by ELISA. Data are expressed as mean ± SEM values. ns: non-significant, * p < 0.05, ** p < 0.01, *** p < 0.001, as compared with untreated groups.

Article Snippet: Antibodies: anti-IL-1β (NB600-633, Novus Biologicals, Littleton, CO, USA); anti-HA (Ab137838, Abcam, Cambridge, UK); anti-Flag (F1804, Sigma-Aldrich, St Louis, MO, USA); anti-NLRP3 (AG-20B-0014-C100, Adipogen, San Diego, CA, USA); anti-ASC (AG-25B-0006-C100, Adipogen, San Diego, CA, USA) Reagents: recombinant human IL-1β (IL038, Sigma-Aldrich, St Louis, MO, USA), recombinant human TNF-α (ab9642, Abcam, Cambridge, UK); Z-YVAD-FMK (S8507, Selleck, Shanghai, China); Z-VAD-FMK (S7023, Selleck, Shanghai, China); MCC950 (S8930, Selleck, Shanghai, China); Bay 11-7082 (S2913, Selleck, Shanghai, China); IL-1R antagonist (sc-221747, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Activation Assay, Derivative Assay, Quantitative RT-PCR, Transfection, Luciferase, Plasmid Preparation, Activity Assay, Positive Control, Enzyme-linked Immunosorbent Assay

Journal: Cell Death & Disease

Article Title: YOD1 protects against MRSA sepsis-induced DIC through Lys33-linked deubiquitination of NLRP3

doi: 10.1038/s41419-024-06731-5

Figure Lengend Snippet: Yod1 −/− mice were pretreated with MCC950 for 30 min, before intravenous injection of MRSA. Mice were sacrificed at 12 h after MRSA administration. The levels of blood markers of DIC (APTT ( A ), D-Dimer ( B ), platelet ( C ), FIB ( D ), TAT ( E ), and PAI-1 ( F )) were assayed. G Representative images of H&E staining of the major organs (liver, lung and kidney) from mice in each group. Scale bar, 50 μm. H The 14-day survival rate was observed after MCC950 treatment ( n = 8). Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: MCC950 Sodium (S7809) was purchased from Selleck (TX, USA).

Techniques: Injection, Staining

Journal: Journal for Immunotherapy of Cancer

Article Title: Targeting the NLRP3 inflammasome abrogates cardiotoxicity of immune checkpoint blockers

doi: 10.1136/jitc-2024-010127

Figure Lengend Snippet: NLRP3 inhibition attenuates ICI-induced cardiac injury. Mice were inoculated with B16F10 melanoma cells on day 0, and injected with either IgG+vehicle, or αPD-1 antibody+vehicle, or αPD-1 antibody+MCC950 every other day from day 7 to day 19. Hearts were collected on day 20. ( A ) Experimental design. ( B ) ELISA for plasma cTnT levels of indicated groups. ( C ) Echocardiographic analysis showing LVEF, LVFS, LVESV, LVEDV, LVIDs, and LVIDd, in mice of each group. ( D, E and F ) Representative immunofluorescence images for CD8 ( D ), F4/80 ( E ), TUNEL ( F ) and their statistical analysis of per high-power field (HPF, average of 3–6 different 200× visual fields) in the hearts of each group; scale bar, 50 µm. ( G ) Representative Masson staining images in mouse hearts of each group and their statistical analysis. Scale bar, 50 µm. ( H ) RT-qPCR analysis for the expression of Il1b , Il6 , Tnf , Ifng , Ccl2 , Ccl3 , Ccl5 . n=8 per group. Data are presented as the mean±SD. Data were analyzed by one-way ANOVA followed by Tukey post hoc multicomparison test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 compared with indicated group.cTnT, cardiac troponin t; ICI, immune checkpoint inhibitor; LVEF, left ventricular ejection fraction; LVFS, left ventricular fractional shortening; LVEDV, left ventricular end-diastolic volume; LVESV, left ventricular end-systolic volume; LVIDd, left ventricular internal dimension in diastole; LVIDs, left ventricular internal dimension in systole; RT-qPCR, reverse transcription quantitative polymerase chain reaction; TUNEL, TdT-mediated dUTP nick-end labeling.

Article Snippet: For NLRP3 blockade treatment, MCC950 (TargetMol, #T6887) was dissolved in saline and administered orally to mice at a dose of 20 mg/kg at specified times.

Techniques: Inhibition, Injection, Enzyme-linked Immunosorbent Assay, Immunofluorescence, TUNEL Assay, Staining, Quantitative RT-PCR, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, End Labeling

Journal: Journal for Immunotherapy of Cancer

Article Title: Targeting the NLRP3 inflammasome abrogates cardiotoxicity of immune checkpoint blockers

doi: 10.1136/jitc-2024-010127

Figure Lengend Snippet: NLRP3 blockade promotes antitumor immunity. (A) Tumor volume measurements of the female B16F10 tumor-bearing mice treated with IgG antibody+vehicle, or IgG+MCC950, or αPD-1 antibody+vehicle, or αPD-1 antibody+MCC950. ( B ) Tumor volume at day 19 of each group. ( C ) Tumor weight at day 20 of each group. ( D ) Flow cytometry analysis for the percentage of myeloid cells, TAM, granulocytic MDSC, and monocytic MDSC in the CD45 + immune cells isolated from the tumor. ( E ) Flow cytometry analysis for the percentage of CD8 + T cells in the CD45 + immune cells, as well as the percentage of CD44 + , IFN-γ and TNF-α-producing cells among CD8 + T cells. n=8 per group. Data are presented as mean±SD. Data were analyzed by one-way ANOVA followed by Tukey post hoc multicomparison test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 compared with indicated group. MDSC, myeloid-derived suppressor cell; TAM, tumor-associated macrophage.

Article Snippet: For NLRP3 blockade treatment, MCC950 (TargetMol, #T6887) was dissolved in saline and administered orally to mice at a dose of 20 mg/kg at specified times.

Techniques: Flow Cytometry, Isolation, Derivative Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Targeting the NLRP3 inflammasome abrogates cardiotoxicity of immune checkpoint blockers

doi: 10.1136/jitc-2024-010127

Figure Lengend Snippet: ScRNA-seq of cardiac immune cells and macrophage cell clusters after MCC950 treatment. (A) UMAP plot showing cell clusters of 68 058 cells by scRNA-seq analysis of mouse hearts from IgG group, αPD-1 group and αPD-1 + MCC950 group. ( B ) Pie chart showing the percentage of each cell cluster in eight immune cell clusters. ( C ) Proportion of each immune cell cluster in different samples. ( D ) The UMAP plot of four macrophage subpopulations. ( E ) Bar plot showing upregulated Gene Ontology biological processes in each macrophage subpopulation. ( F ) Dot plot showing the expression of specific genes in each macrophage subpopulation. ( G ) Proportion of each macrophage subpopulation in different samples. ( H ) Bar plot showing the differences in GO biological processes. DCs, dendritic cells; ILCs, innate lymphocytes; RBCs, red blood cells; ScRNA-seq, single-cell RNA sequencing.

Article Snippet: For NLRP3 blockade treatment, MCC950 (TargetMol, #T6887) was dissolved in saline and administered orally to mice at a dose of 20 mg/kg at specified times.

Techniques: Expressing, RNA Sequencing Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Targeting the NLRP3 inflammasome abrogates cardiotoxicity of immune checkpoint blockers

doi: 10.1136/jitc-2024-010127

Figure Lengend Snippet: NLRP3 inhibition ameliorates cardiotoxicity of combined ICI therapy. Mice were inoculated with B16F10 melanoma cells on day 0, and were administrated with IgG+vehicle, or anti-PD-1 + anti-CTLA-4 + vehicle, or anti-PD-1 + anti-CTLA-4 + MCC950 every other day from day 7 to day 19. Hearts were collected on day 20. ( A ) Experimental design. ( B ) ELISA for plasma cTnT levels of IgG group, dual ICI (D-ICI) group and dual ICI+MCC950 (D-ICI+MCC950) group. ( C ) Echocardiographic analysis showing cardiac function of each group. ( D , E , and F ) Representative immunofluorescence images for CD8 ( D ), F4/80 ( E ), TUNEL ( F ) and their statistical analysis of per high-power field (HPF, average of 3–6 different 200× visual fields) in the heart of each group; scale bar, 50 µm. ( G ) Representative Masson staining images in mouse hearts of each group and their statistical analysis. Scale bar, 50 µm. ( H ) RT-qPCR analysis for relative mRNA expression of Il1b , Il6 , Tnf , Ifng , Ccl2 , Ccl3 , Ccl5 in the heart of each group. ( I ) Tumor volume measurements of the female B16F10 tumor-bearing mice of each group. ( J ) Tumor volume at day 19 of each group (left) and tumor weight at day 20 of each group (right). n=8 per group. Data are presented as mean±SD. Data were analyzed by one-way ANOVA followed by Tukey post hoc multicomparison test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 compared with indicated group. RT-qPCR, reverse transcription quantitative polymerase chain reaction; TUNEL, TdT-mediated dUTP nick-end labeling.

Article Snippet: For NLRP3 blockade treatment, MCC950 (TargetMol, #T6887) was dissolved in saline and administered orally to mice at a dose of 20 mg/kg at specified times.

Techniques: Inhibition, Enzyme-linked Immunosorbent Assay, Immunofluorescence, TUNEL Assay, Staining, Quantitative RT-PCR, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, End Labeling

Journal: Veterinary Research

Article Title: Staphylococcus aureus mediates pyroptosis in bovine mammary epithelial cell via activation of NLRP3 inflammasome

doi: 10.1186/s13567-022-01027-y

Figure Lengend Snippet: NLRP3 inflammasome regulates ASC speck formation during S. aureus . Fluorescence microscopy images of MAC-T cells immunoassayed for ASC (red) with or without MCC950 after 4 h of S. aureus treatment.

Article Snippet: Caspase-1 inhibitor VX765 and NLRP3 inflammasome inhibitor MCC950 were obtained from Glpbio (USA) and used at final concentrations of 100 and 15 μM.

Techniques: Fluorescence, Microscopy

Journal: Veterinary Research

Article Title: Staphylococcus aureus mediates pyroptosis in bovine mammary epithelial cell via activation of NLRP3 inflammasome

doi: 10.1186/s13567-022-01027-y

Figure Lengend Snippet: NLRP3 inflammasome activation by S. aureus is essential for the generation of GSDMD-N and the release of IL-1β and IL-18. A Activated caspase-1 and B GSDMD-N or released C IL-1β and D IL-18 of MAC-T cells treated with or without NLRP3 inhibitor MCC950 or caspase-1 inhibitor VX765 for 1.5 h prior to treatment with S. aureus for 4 h. E Levels of IL-1β and F IL-18 released after S. aureus treatment of MAC-T cells for 4 h in the presence or absence of 25, 50, or 75 mM KCl.

Article Snippet: Caspase-1 inhibitor VX765 and NLRP3 inflammasome inhibitor MCC950 were obtained from Glpbio (USA) and used at final concentrations of 100 and 15 μM.

Techniques: Activation Assay