Journal: PLoS ONE

Article Title: Pepsin Digest of Wheat Gliadin Fraction Increases Production of IL-1β via TLR4/MyD88/TRIF/MAPK/NF-κB Signaling Pathway and an NLRP3 Inflammasome Activation

doi: 10.1371/journal.pone.0062426

Figure Lengend Snippet: IL-1β (A), IL-18 (B) and IL-1α (C) levels were quantified in cell supernatants by ELISA. Data are given as mean ± SD from 39 patients and 15 healthy donors (HD). *P<0.05, **P<0.01 (CD vs. HD). ( D ). PDWGF-induced activation of PBMC is not due to LPS contamination. Results are shown as the percentage of the cytokine production from 4 CD patients. The data were normalized to the result from untreated cells which was set as 100%. Mean ± SD, n = 4 independent experiments, ***P<0.001 compared to untreated cells.

Article Snippet: Anti-human IL-1 β/IL-1F2 Ab and anti-mouse IL-1β/IL-1F2 Ab were from R&D Systems (Minneapolis, MN, USA), while anti-human cleaved IL-1β Ab was purchased from Cell Signaling Technology.

Techniques: Enzyme-linked Immunosorbent Assay, Activation Assay

Journal: PLoS ONE

Article Title: Pepsin Digest of Wheat Gliadin Fraction Increases Production of IL-1β via TLR4/MyD88/TRIF/MAPK/NF-κB Signaling Pathway and an NLRP3 Inflammasome Activation

doi: 10.1371/journal.pone.0062426

Figure Lengend Snippet: ( A ) Caspase-1 inhibitor Z-YVAD-fmk reduced PDWGF-induced IL-1β, but not IL-1α or TNF-α production. Mean ± SD, n = 10 independent experiments, ***P<0.001 compared to PDWGF-treated cells. ( B ) Western blot analysis for the expression of pro-IL-1β and mature IL-1β and ( C ) for caspase-1 and caspase-1 p10 in cell lysates (CL) and cell culture supernatants (CS) from PBMC. Representative blots from 5 independent experiments are shown. β-actin was used as a loading control. ( D ) The fold increase (FI) (densitometry analysis) of the quantity of caspase-1 p10 normalized to non-activated cells. Mean ± SD, n = 5 independent experiments, *P<0.05 compared to non-activated cells. ( E, F ) Direct activation of caspase-1 in PDWGF-treated PBMC, assessed by flow cytometry using a cell-permeable fluorescent probe. Results are shown as ( E ) a representative histogram from 2 CD patients and 1 HD; and ( F ) as the percentage of the MFI from 12 CD patients and 10 HD. The data were normalized to the result from untreated cells, which was set as 100%. Mean ± SD, *P<0.05 compared to untreated cells. CD, celiac disease patients; HD, healthy donors.

Article Snippet: Anti-human IL-1 β/IL-1F2 Ab and anti-mouse IL-1β/IL-1F2 Ab were from R&D Systems (Minneapolis, MN, USA), while anti-human cleaved IL-1β Ab was purchased from Cell Signaling Technology.

Techniques: Western Blot, Expressing, Cell Culture, Activation Assay, Flow Cytometry

Journal: PLoS ONE

Article Title: Pepsin Digest of Wheat Gliadin Fraction Increases Production of IL-1β via TLR4/MyD88/TRIF/MAPK/NF-κB Signaling Pathway and an NLRP3 Inflammasome Activation

doi: 10.1371/journal.pone.0062426

Figure Lengend Snippet: Representative blots from 5 independent experiments are shown. ( C ) Inhibition of ROS modulate PDWGF-induced IL-1β secretion, mean ± SD, n = 10; as well as (D) pro-IL-1β production from PBMC of CD patients. Representative blots from 3 independent experiments are shown. β-actin was used as a loading control. ***P<0.001 vs. PDWGF-treated cells.

Article Snippet: Anti-human IL-1 β/IL-1F2 Ab and anti-mouse IL-1β/IL-1F2 Ab were from R&D Systems (Minneapolis, MN, USA), while anti-human cleaved IL-1β Ab was purchased from Cell Signaling Technology.

Techniques: Inhibition

Journal: PLoS ONE

Article Title: Pepsin Digest of Wheat Gliadin Fraction Increases Production of IL-1β via TLR4/MyD88/TRIF/MAPK/NF-κB Signaling Pathway and an NLRP3 Inflammasome Activation

doi: 10.1371/journal.pone.0062426

Figure Lengend Snippet: PDWGF-primed PBMC were treated with SB203580 (SB), SP600125 (SP), UO125 (UO), TPCK, or PDWGF, alone or in combination for 24 h. ( A ) IL-1β was quantified in cell supernatants by ELISA. Mean ± SD, n = 5 independent experiments. ***P<0.001 vs. PDWGF-treated cells. ( B ) Pro-IL-1β levels were examined in cell lysates by immunoblotting from 5 experiments. β-actin was used as a loading control.

Article Snippet: Anti-human IL-1 β/IL-1F2 Ab and anti-mouse IL-1β/IL-1F2 Ab were from R&D Systems (Minneapolis, MN, USA), while anti-human cleaved IL-1β Ab was purchased from Cell Signaling Technology.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot

Journal: PLoS ONE

Article Title: Pepsin Digest of Wheat Gliadin Fraction Increases Production of IL-1β via TLR4/MyD88/TRIF/MAPK/NF-κB Signaling Pathway and an NLRP3 Inflammasome Activation

doi: 10.1371/journal.pone.0062426

Figure Lengend Snippet: ( A ) BMDC from WT, NLRP3−/− and ASC−/− mice were exposed to PDWGF (100 µg/ml) alone for 24 h; or first PDWGF was added for 21.5 h, the subsequently ATP (2 mM) was added for additional 2.5 h. IL-1β was measured in culture supernatants. ( B ) Flow-cytometric evaluation of PDWGF-induced maturation assessed by CD40, CD80, and CD86 expression on BMDC from WT and NLRP3−/− mice. WT and NLRP3−/− BMDC were cultured with 100 µg/ml of PDWGF (green), as well as 0.1 µg/ml of LPS (red) or 100 µg/ml of OVA (grey-filled) as positive and negative controls, respectively. Isotype controls are represented in black overlays. ( C ) Cells were preincubated with caspase-1 inhibitor Z-YVAD-fmk for 30 min, and then exposed to PDWGF in combination with ATP. Production of IL-1β was measured in culture supernatants. Results are expressed as mean ± SD from 4 independent experiments. The levels of significance for KO BMDC vs. WT BMDC are indicated as follows: *P<0.05, **P<0.01, and ***P<0.001.

Article Snippet: Anti-human IL-1 β/IL-1F2 Ab and anti-mouse IL-1β/IL-1F2 Ab were from R&D Systems (Minneapolis, MN, USA), while anti-human cleaved IL-1β Ab was purchased from Cell Signaling Technology.

Techniques: Expressing, Cell Culture

Journal: PLoS ONE

Article Title: Pepsin Digest of Wheat Gliadin Fraction Increases Production of IL-1β via TLR4/MyD88/TRIF/MAPK/NF-κB Signaling Pathway and an NLRP3 Inflammasome Activation

doi: 10.1371/journal.pone.0062426

Figure Lengend Snippet: WT BMDC and MyD88−/−, TRIF−/−, and IL-1R−/− KO BMDC were treated with PDWGF alone or in combination with ATP and ( A ) IL-1β production was evaluated after 24 h. ( B ) Cell lysates were evaluated for de novo synthesis of pro-IL-1β ( C ) WT BMDC and TLR2−/−, TLR4−/−, and TLR2/4−/− KO BMDC were treated with PDWGF alone or in combination with ATP, and IL-1β production was evaluated after 24 h. ( D ) Cell lysates were evaluated for de novo synthesis of pro-IL-1β. Data in (A) and (C) are expressed as mean ± SD from 5 independent experiments. *P<0.05, **P<0.01 vs. WT BMDC. Blots in (B) and (D) are representative from 3 independent experiments. β-actin was used as a loading control. ( E ) Celiac PBMC were treated with PDWGF alone or in combination with anti-TLR4 or anti-TLR2 Ab. IL-1β secretion was evaluated after 24 h. LPS was used as a positive control. Mean ± SD, 8 independent experiments. **P<0.01, ***P<0.001 vs. cells without anti-TLR Ab.

Article Snippet: Anti-human IL-1 β/IL-1F2 Ab and anti-mouse IL-1β/IL-1F2 Ab were from R&D Systems (Minneapolis, MN, USA), while anti-human cleaved IL-1β Ab was purchased from Cell Signaling Technology.

Techniques: Positive Control

Journal: PLoS ONE

Article Title: Pepsin Digest of Wheat Gliadin Fraction Increases Production of IL-1β via TLR4/MyD88/TRIF/MAPK/NF-κB Signaling Pathway and an NLRP3 Inflammasome Activation

doi: 10.1371/journal.pone.0062426

Figure Lengend Snippet: PDWGF as well as α-amylase inhibitor (non-treated or R/A treated) were added to celiac PBMC. IL-1β secretion was evaluated after 24 h. Results are shown as the percentage of the cytokine production from 5 CD patients. The data were normalized to the result from PDWGF-treated cells which was set as 100%. Mean ± SD, 5 independent experiments. ***P<0.001 vs. non-treated PDWGF.

Article Snippet: Anti-human IL-1 β/IL-1F2 Ab and anti-mouse IL-1β/IL-1F2 Ab were from R&D Systems (Minneapolis, MN, USA), while anti-human cleaved IL-1β Ab was purchased from Cell Signaling Technology.

Techniques:

Journal: Frontiers in Immunology

Article Title: Internalization of the Membrane Attack Complex Triggers NLRP3 Inflammasome Activation and IL-1β Secretion in Human Macrophages

doi: 10.3389/fimmu.2021.720655

Figure Lengend Snippet: The Membrane Attack Complex induces inflammasome activation in human macrophages. (A–K) THP1 cells (A–D) and human MDMs (E–K) were treated with vehicles or primed with 1 µg/mL LPS for 3 hrs followed by stimulation with 10 µM nigericin (Nig) for 45 mins or with 10 µg/mL anti-CD59 mAb, 10 µg/mL C5b6, unless otherwise specified, C7, C8 and C9 (MAC) for 3 hrs. (A, E, J) IL-1β secretion was measured by ELISA. (B, F) IL-1β cleavage was analyzed in supernatants by immunoblot. (C) GSDMD processing was analyzed in lysates by immunoblot. (D, H, K) LDH release was measured as a proxy for cell death. (F) Pro-IL-1β production was analyzed in supernatants and cell lysates by immunoblot. (G) Caspase 1 activity was measured in cell supernatants and expressed as fold increase vs untreated cells. (I) TCC (MAC) amount in cell lysates was measured by ELISA. (A, D, E, G–K) Data is plotted as mean ± SD and is representative of three (D, G, I–K) , six (A) , seven (E) or eight (H) independent experiments. (E, G–K) Each color represents a matched donor. (A, D) Each dot represents a repeat. (A, D, E, G–K) Statistical significance was measured by one-way ANOVA (A, D, G–K) or Friedman test (E) (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). (B , C , F) Blots are representative of two independent experiments with similar results.

Article Snippet: The primary antibodies used for immunoblotting and their final concentrations were goat Ab anti-human IL‐1β (0.1 µg/mL, R&D Systems), rabbit Ab anti-human GSDMD (0.14 µg/mL, Novus Biologicals), rabbit mAb anti‐human caspase‐1 (1:1000, D7F10, Cell Signalling Technology), mouse mAb anti‐human NLRP3 (1 μg/mL, Cryo-2, Adipogen), mouse mAb anti‐β‐actin‐HRP (0.2 μg/mL, AC-15, Sigma).

Techniques: Membrane, Activation Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Activity Assay

Journal: Frontiers in Immunology

Article Title: Internalization of the Membrane Attack Complex Triggers NLRP3 Inflammasome Activation and IL-1β Secretion in Human Macrophages

doi: 10.3389/fimmu.2021.720655

Figure Lengend Snippet: MAC-mediated IL-1β secretion is dependent on NLRP3 and GSDMD. (A–H) Human MDMs (A, B, F–H) and wild-type THP1 or THP1 nlrp3-/- (C–E) cells were treated with vehicles or primed with 1 µg/mL LPS for 3 hrs followed by stimulation with 10 µM nigericin (Nig) for 45 mins or with 10 µg/mL anti-CD59 mAb, C5b6, C7, C8 and C9 (MAC) for 3 hrs. (A, B, F–H) MAC treatment was carried out in the presence or absence of 1 µM MCC950 (A, B) or 10 µM NSA (F–H) . (A, D, G) IL-1β secretion was measured by ELISA. (B, E, H) LDH release was measured as a proxy for cell death. (C, F) NLRP3 (C) and GSDMD (F) production were analyzed in cell lysates by immunoblot. (A, B, D, E, G, H) Data is plotted as mean ± SD and is representative of five (A, B) or three (D, E, G, H) independent experiments. (A, B, G, H) Each color represents a matched single donor. (D, E) Each color represents a repeat. (A, B, D, E, G, H) Statistical significance was measured by Friedman test (A) or one-way ANOVA (B, D, E, G, H) (*p < 0.05, **p < 0.01, ***p < 0.001). (C, F) Blots are representative of two independent experiments with similar results.

Article Snippet: The primary antibodies used for immunoblotting and their final concentrations were goat Ab anti-human IL‐1β (0.1 µg/mL, R&D Systems), rabbit Ab anti-human GSDMD (0.14 µg/mL, Novus Biologicals), rabbit mAb anti‐human caspase‐1 (1:1000, D7F10, Cell Signalling Technology), mouse mAb anti‐human NLRP3 (1 μg/mL, Cryo-2, Adipogen), mouse mAb anti‐β‐actin‐HRP (0.2 μg/mL, AC-15, Sigma).

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Frontiers in Immunology

Article Title: Internalization of the Membrane Attack Complex Triggers NLRP3 Inflammasome Activation and IL-1β Secretion in Human Macrophages

doi: 10.3389/fimmu.2021.720655

Figure Lengend Snippet: The MAC triggers inflammasome assembly and localizes to the NLRP3-ASC speck. (A–F) Human MDMs (A–E) and THP1 nlrp3-/- /eGFP-nlrp3 cells (F) were treated with vehicles or primed with 1 µg/mL LPS for 3 hrs followed by stimulation with 10 µg/mL anti-CD59 mAb, C5b6, C7, C8 and C9 labelled with JF-549 (MAC) for the indicated time (A–C) or 3hrs (D–F) . (A) Representative confocal images of C9 (red) and ASC (cyan) overtime. (B) Percentage of cells with ASC specks over time. (C) Percentage of cells with C9 over time. (D) Confocal imaging of C9 (red), ASC (cyan) and TCC (green). (E) Percentage of ASC speck positive cells with C9 in the speck. (F) Confocal imaging of C9 (red), ASC (cyan) and NLRP3 (green). (A, D, F) Dashed lines represent the outline of the cell, arrows point to specks, magenta lines correspond to the plotted line profiles and scale bars are 10 µm (A) and 5 µm (D, F) . (B, C, E) Data is plotted as mean ± SD, each color represents a matched donor. (B, C) Statistical significance was measured by one-way ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). (A–F) Data is representative of 3 independent experiments.

Article Snippet: The primary antibodies used for immunoblotting and their final concentrations were goat Ab anti-human IL‐1β (0.1 µg/mL, R&D Systems), rabbit Ab anti-human GSDMD (0.14 µg/mL, Novus Biologicals), rabbit mAb anti‐human caspase‐1 (1:1000, D7F10, Cell Signalling Technology), mouse mAb anti‐human NLRP3 (1 μg/mL, Cryo-2, Adipogen), mouse mAb anti‐β‐actin‐HRP (0.2 μg/mL, AC-15, Sigma).

Techniques: Imaging

Journal: Frontiers in Immunology

Article Title: Internalization of the Membrane Attack Complex Triggers NLRP3 Inflammasome Activation and IL-1β Secretion in Human Macrophages

doi: 10.3389/fimmu.2021.720655

Figure Lengend Snippet: Internalization of the MAC is required for inflammasome activation. (A, B) LPS-primed human MDMs were treated with 10 µg/mL anti-CD59 mAb, C5b6, C7, C8 and C9 labelled with AF-647 (MAC) for the indicated time. (A) Confocal imaging of EEA1(green) and C9 (red). Yellow squares represent zoom regions and scale bars are 10 µm in full cell pictures and 1 µm in zoom regions. (B) Pearson’s correlation coefficient of C9 compared to EEA1. (C–G) Human MDMs were treated with vehicles or primed with 1 µg/mL LPS for 3 hrs followed by stimulation with 10 µg/mL anti-CD59 mAb, C5b6, C7, C8 and C9 for 3 hrs (C–E) or 10 µM nigericin (Nig) for 45 mins (F, G) in the presence or absence of 0.1 µg/mL nystatin (Nys), 10 µM cytochalasin D (Cyt) or 10 µM dyanasore (Dyn). (C, F) IL-1β secretion was measured by ELISA. (D, G) LDH release was measured as a proxy for cell death. (E) GSDMD processing was analyzed in cell lysates by immunoblot. (C, D, F, H) Data is plotted as mean ± SD, each color represents a matched donor and statistical significance was measured by one-way ANOVA (B–D, G) or Friedman’s test (F) (*p < 0.05, **p < 0.01, ***p < 0.001). (A–G) Data is representative of 3 (A–E) or 5 (F, G) independent experiments.

Article Snippet: The primary antibodies used for immunoblotting and their final concentrations were goat Ab anti-human IL‐1β (0.1 µg/mL, R&D Systems), rabbit Ab anti-human GSDMD (0.14 µg/mL, Novus Biologicals), rabbit mAb anti‐human caspase‐1 (1:1000, D7F10, Cell Signalling Technology), mouse mAb anti‐human NLRP3 (1 μg/mL, Cryo-2, Adipogen), mouse mAb anti‐β‐actin‐HRP (0.2 μg/mL, AC-15, Sigma).

Techniques: Activation Assay, Imaging, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Frontiers in Immunology

Article Title: Internalization of the Membrane Attack Complex Triggers NLRP3 Inflammasome Activation and IL-1β Secretion in Human Macrophages

doi: 10.3389/fimmu.2021.720655

Figure Lengend Snippet: Endocytosis of the MAC triggers disruption of the TGN. (A–D) Human MDMs were treated with vehicles or primed with 1 µg/mL LPS for 3 hrs followed by stimulation with 10 µg/mL anti-CD59 mAb, C5b6, C7, C8 and C9 labelled with AF-647 (MAC) for the indicated time or nigericin (Nig) for 45 mins. (E, F) Human MDMs were treated with vehicles or primed with LPS and the MAC for 3 hrs in the presence or absence of 0.1 µg/mL nystatin (Nys) or 1 µM MCC950. (A) Representative confocal images of the different TGN conformations: intact TGN (iTGN), fragmented TGN (fTGN) and dispersed TGN (dTGN). (B, E) Percentage of cells with dTGN. (C) Representative confocal images of the results analyzed in panel (B) . (F) Representative confocal images of the results analyzed in panel (E) . (C, F) TGN46 (green) and C9 (red) and brightfield images (gray). Dashed lines represent cell outlines and scale bars are 10 µm. (D) Pie charts representing the percentage of cells with dTGN, fTGN and iTGN in each condition. (B, E) Data is plotted as mean ± SD, each color represents a matched donor and statistical significance was measured by Friedman’s test (*p < 0.05, **p < 0.01, ***p < 0.001). (B–E) Data is representative of 3 (E, F) or 4 (B–D) independent experiments.

Article Snippet: The primary antibodies used for immunoblotting and their final concentrations were goat Ab anti-human IL‐1β (0.1 µg/mL, R&D Systems), rabbit Ab anti-human GSDMD (0.14 µg/mL, Novus Biologicals), rabbit mAb anti‐human caspase‐1 (1:1000, D7F10, Cell Signalling Technology), mouse mAb anti‐human NLRP3 (1 μg/mL, Cryo-2, Adipogen), mouse mAb anti‐β‐actin‐HRP (0.2 μg/mL, AC-15, Sigma).

Techniques: Disruption

Journal: Frontiers in Immunology

Article Title: Internalization of the Membrane Attack Complex Triggers NLRP3 Inflammasome Activation and IL-1β Secretion in Human Macrophages

doi: 10.3389/fimmu.2021.720655

Figure Lengend Snippet: A proposed model for MAC-mediated NLRP3 inflammasome activation in human macrophages. Formation of the MAC in the cell membrane triggers its internalization via EEA1+ endosomes. Endocytosis of the MAC leads to dispersion of the trans-Golgi network (TGN) and to the assembly of the NLRP3 inflammasome. The roles of TGN dispersion as well as any direct involvement of MAC components on inflammasome assembly remain to be elucidated. Within the NLRP3 complex, caspase 1 (Casp-1) becomes active and cleaves GSDMD into its pore-forming subunit, NT-GSDMD, and pro-IL-1β into its active form IL-1β. Multiple NT-GSDMD subunits assemble in the cell membrane to form GSDMD pores resulting in the release of IL-1β to the extracellular milieu.

Article Snippet: The primary antibodies used for immunoblotting and their final concentrations were goat Ab anti-human IL‐1β (0.1 µg/mL, R&D Systems), rabbit Ab anti-human GSDMD (0.14 µg/mL, Novus Biologicals), rabbit mAb anti‐human caspase‐1 (1:1000, D7F10, Cell Signalling Technology), mouse mAb anti‐human NLRP3 (1 μg/mL, Cryo-2, Adipogen), mouse mAb anti‐β‐actin‐HRP (0.2 μg/mL, AC-15, Sigma).

Techniques: Activation Assay, Membrane, Dispersion