Journal: Cell Reports Medicine

Article Title: Truncating NFKB1 variants cause combined NLRP3 inflammasome activation and type I interferon signaling and predispose to necrotizing fasciitis

doi: 10.1016/j.xcrm.2024.101503

Figure Lengend Snippet: Truncating mutations of NFKB1 cause enhanced NLRP3 inflammasome activation and aberrant gene expression in patient-derived macrophages (A) Schematic representation of p105/p50 protein domains and sites of mutations. Conserved Rel homology domain includes sequences needed for nuclear location, binding to DNA, and dimer formation with Rel family proteins and inhibitor proteins. GRR, glycine-rich region. (B and C) HMDMs (B) or PBMCs (C) were stimulated with LPS for indicated times and NFKB1 (B) mRNA or (C) protein expression of p105 and p50 was analyzed by RT-qPCR or western blot. Quantifications, blots, and total protein loading (TPL) are shown. The data are shown as mean ± SD. Healthy control 2 (indicated with the arrowhead) is included twice to enable comparison between the blots. (D and E) HMDMs were stimulated with (D) LPS and ATP or (E) monosodium urate crystals (MSUs) and secretion of mature IL-1β was detected by ELISA. Average of 10 separately processed healthy volunteers is also shown. (F and G) HMDMs were activated with LPS or LPS and LPS-binding protein (LBP) for indicated times. Gene expression and secretion of IFN-β were analyzed by RT-qPCR (left) and ELISA (right) (F) and expressions of IFIT2 , CXCL10 , and IL10 were analyzed by RT-qPCR (G). (B) 3 controls, 4 variant carriers; (C) 5 controls, 6 variant carriers; (D) 15 controls, 5 variant carriers; (E) 15 controls, 4 variant carriers; (F) RT-qPCR: 3 controls, 5 variant carriers, ELISA: 1 control, 3 variant carriers; (G) 3 controls, 5 variant carriers. See also Figures S1 and .

Article Snippet: Specific inhibitor of mitochondrial reactive oxygen species (MitoTEMPO, Sigma SML0703) 200 μM, and NLRP3 inflammasome (MCC950, Invivogen inf-mcc) 10 μM, were applied 1 h before ATP activation (45 min) for HMDMs and THP-1 monocytes.

Techniques: Activation Assay, Expressing, Derivative Assay, Binding Assay, Quantitative RT-PCR, Western Blot, Control, Comparison, Enzyme-linked Immunosorbent Assay, Variant Assay

Journal: Cell Reports Medicine

Article Title: Truncating NFKB1 variants cause combined NLRP3 inflammasome activation and type I interferon signaling and predispose to necrotizing fasciitis

doi: 10.1016/j.xcrm.2024.101503

Figure Lengend Snippet: NLRP3 inflammasome activation is proportional to expression of NFKB1 (A) NF-κB1 basal expression in NFKB1 R157∗/R157∗ , NFKB1 −/− , or NFKB1 −/+ and WT or mock THP-1 monocyte lysates. Blot and TPL are shown. (B) NFKB1 R157∗/R157∗ and WT THP-1 monocytes were stimulated with LPS and ATP or nigericin. Secreted mature IL-1β was detected by ELISA. (C) NFKB1 R157∗/R157∗ and WT THP-1 monocytes were activated with LPS for indicated times. Expression of IFNB1 , IFIT2 , and CXCL10 was analyzed by RT-qPCR. (D) NFKB1 R157∗/R157∗ and WT THP-1 monocytes were stimulated with LPS and treated with caspase-1/4 inhibitor (Z-YVAD-FMK) 1 h before ATP. Secreted mature IL-1β was detected by ELISA. (E) NFKB1 R157∗/R157∗ and WT THP-1 monocytes were stimulated with LPS and treated with specific NLRP3 inhibitor (MCC950) 1 h before ATP. Secreted mature IL-1β was detected by ELISA. (F) Immunoblot analysis of NLRP3 immunoprecipitated with ASC and immunoblots of ASC and NLRP3 from lysates of NFKB1 R157∗/R157∗ and WT THP-1 monocytes primed with LPS and activated with ATP or left untreated. Quantifications, representative blots, and TPL are shown. (G and H) NFKB1 R157∗/R157∗ , NFKB1 −/− , mock, and WT THP-1 monocytes (G) or NFKB1 −/− , NFKB1 −/+ , and mock THP-1 monocytes (H) were activated with LPS and ATP. Secreted mature IL-1β was detected by ELISA. (I) NFKB1 R157∗/R157∗ and WT THP-1 monocytes were stimulated with LPS or Pam3Cys-SKKKK or transfected with low-molecular-weight poly(I:C) to activate RIG-I/MDA-5. Expressions of IFNB1 and IFIT2 were analyzed by RT-qPCR. Two-way ANOVA (B–E and G–I) or two-way repeated measures (RM) ANOVA (F) followed by Tukey’s (G and H) or Šidak’s (B–F and I) tests. The data are shown as mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. (A) WT, NFKB1 −/− , and mock n = 2, NFKB1 −/− and NFKB1 −/+ n = 4; (B–I) n = 4. See also Figures S2 and .

Article Snippet: Specific inhibitor of mitochondrial reactive oxygen species (MitoTEMPO, Sigma SML0703) 200 μM, and NLRP3 inflammasome (MCC950, Invivogen inf-mcc) 10 μM, were applied 1 h before ATP activation (45 min) for HMDMs and THP-1 monocytes.

Techniques: Activation Assay, Expressing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Western Blot, Immunoprecipitation, Transfection, Molecular Weight

Journal: Cell Reports Medicine

Article Title: Truncating NFKB1 variants cause combined NLRP3 inflammasome activation and type I interferon signaling and predispose to necrotizing fasciitis

doi: 10.1016/j.xcrm.2024.101503

Figure Lengend Snippet: Truncating variants of NFKB1 impair autophagy (A) MitoSOX staining of untreated NFKB1 R157∗/R157∗ and WT THP-1 monocytes was analyzed by flow cytometry. Quantifications and representative plots are shown. (B) HMDMs (left) or NFKB1 R157∗/R157∗ and WT THP-1 monocytes (right) were activated with LPS, and MitoTempo was applied 1 h before ATP. Secreted mature IL-1β was detected by ELISA. (C) ρ 0 or conventional NFKB1 R157∗/R157∗ and WT THP-1 monocytes were activated with LPS and secreted mature IL-1β was detected by ELISA. (D) HMDMs (left) or NFKB1 R157∗/R157∗ and WT THP-1 monocytes (right) were activated with LPS for indicated times and SQSTM1 was blotted from lysates. Quantifications, representative blots, and TPL are shown. (E) NFKB1 R157∗/R157∗ and WT THP-1 monocytes were inhibited with E-64d and pepstatin A (abbreviated as EP) 1 h prior to LPS (for indicated times). LC3 was blotted from cell lysates. Quantifications, representative blot, and TPL are shown. (F and G) NFKB1 R157∗/R157∗ and WT THP-1 monocytes were treated with LPS and (F) 3-MA (6 h 45 min) or (G) rapamycin (17 h 45 min) and ATP (for the last 45 min). Secreted mature IL-1β was detected by ELISA and is expressed as fold change relative to the respective cells stimulated with LPS and ATP. (H) NFKB1 R157∗/R157∗ and WT THP-1 monocytes were activated with LPS (18 h). Cells staining positive for Hoechst (nuclear stain) and autophagic vesicle marker were analyzed by flow cytometry. Quantitation and representative plots are shown. (I) Schematic representation of the effects of truncating NFKB1 mutations on macroautophagy and NLRP3 inflammasome activation. Wilcoxon test (A), one-way RM-ANOVA (B), or two-way ANOVA (C–H) followed by Dunnett’s (B), Šidak’s (D–G), or Tukey’s test (C and H). The data are shown as mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. (A) n = 8; (B) HMDMs: 1 control, 3 variant carriers, THP-1 n = 4; (C–E) THP-1 n = 4; (D) HMDMs: 2 controls, 4 variant carriers; (F–H) THP-1 n = 4. See also Figure S4 .

Article Snippet: Specific inhibitor of mitochondrial reactive oxygen species (MitoTEMPO, Sigma SML0703) 200 μM, and NLRP3 inflammasome (MCC950, Invivogen inf-mcc) 10 μM, were applied 1 h before ATP activation (45 min) for HMDMs and THP-1 monocytes.

Techniques: Staining, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Marker, Quantitation Assay, Activation Assay, Control, Variant Assay

Journal: Cell Reports Medicine

Article Title: Truncating NFKB1 variants cause combined NLRP3 inflammasome activation and type I interferon signaling and predispose to necrotizing fasciitis

doi: 10.1016/j.xcrm.2024.101503

Figure Lengend Snippet: Reduction of autophagy results in accumulation of key proteins of the NLRP3 inflammasome and interferon signaling (A–C) HMDMs (left) and NFKB1 R157∗/R157∗ and WT THP-1 monocytes (right) were activated with LPS for indicated times, and the expression of (A) NLRP3, (B) pro-IL-1β, or (C) TRIF in cell lysates was blotted. (D and E) NFKB1 R157∗/R157∗ and WT THP-1 monocytes were activated with LPS for indicate times and (D) total protein secretion was assessed by silver staining or (E) secretion of SQSTM1 was blotted from supernatant. Quantifications, representative blots, and TPL (A–C) are shown. Two-way ANOVA followed by Šidak’s test (A–E). The data are shown as mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. (A–C) 2 controls, 4 variant carriers; THP-1 n = 4; (D) n = 4; (E) n = 3.

Article Snippet: Specific inhibitor of mitochondrial reactive oxygen species (MitoTEMPO, Sigma SML0703) 200 μM, and NLRP3 inflammasome (MCC950, Invivogen inf-mcc) 10 μM, were applied 1 h before ATP activation (45 min) for HMDMs and THP-1 monocytes.

Techniques: Expressing, Silver Staining, Variant Assay

Journal: Cell Reports Medicine

Article Title: Truncating NFKB1 variants cause combined NLRP3 inflammasome activation and type I interferon signaling and predispose to necrotizing fasciitis

doi: 10.1016/j.xcrm.2024.101503

Figure Lengend Snippet:

Article Snippet: Specific inhibitor of mitochondrial reactive oxygen species (MitoTEMPO, Sigma SML0703) 200 μM, and NLRP3 inflammasome (MCC950, Invivogen inf-mcc) 10 μM, were applied 1 h before ATP activation (45 min) for HMDMs and THP-1 monocytes.

Techniques: Control, Recombinant, Lysis, Protease Inhibitor, Western Blot, Cell Culture, Molecular Weight, Staining, Sequencing, Modification, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, Transfection, Quantitation Assay, Mutagenesis, Software, CRISPR, Variant Assay, Cytometry