Journal: Mucosal immunology

Article Title: Mechanisms of NLRP3 inflammasome activation and its role in NSAID-induced enteropathy.

doi: 10.1038/mi.2015.89

Figure Lengend Snippet: Figure 1 Small intestinal damage and cytokine expression after indomethacin administration. (a) Time course of lesion indices after indomethacin administration. The areas of macroscopically visible lesions, stained with Evans blue (arrows), were measured, summed per small intestine, and used as the lesion index. N ¼ 5. (b–d) The mRNA expression levels of tumor necrosis factor-a (TNF-a), interleukin-1b (IL-1b), and IL-18 as determined by quantitative reverse transcription–PCR. mRNA levels are expressed as ratios relative to the mean value for normal small intestinal tissue from untreated mice. N ¼ 6–8. (e, f) Protein concentrations of IL-1b and IL-18 in the small intestine were measured by enzyme-linked immunosorbent assay. N ¼ 5–8. *Po0.05, **Po0.01 vs. untreated control (0 h group). Data in a–f are representative of three independent experiments.

Article Snippet: In addition, mice were intraperitoneally administered neutralizing goat anti-IL-1b polyclonal antibody or normal goat IgG (both from R&D Systems) at a dose of 100 mg per mouse at 0 and 6 h after indomethacin treatment.

Techniques: Expressing, Staining, Reverse Transcription, Enzyme-linked Immunosorbent Assay, Control

Journal: Mucosal immunology

Article Title: Mechanisms of NLRP3 inflammasome activation and its role in NSAID-induced enteropathy.

doi: 10.1038/mi.2015.89

Figure Lengend Snippet: Figure 2 Activation of interleukin-1b (IL-1b) and caspase-1 (Casp-1) during the development of indomethacin-induced small intestinal damage. Expression levels of IL-1b and Casp-1 in the small intestine were evaluated by western blot analysis. (a) Representative western blots for IL-1b and Casp-1. (b–e) Expression levels of (b) pro-IL-1b, (c) mature IL-1b, (d) pro-Casp-1, and (e) cleaved Casp-1 were normalized to that of b-actin. Data are from a single experiment, representative of at least two independent experiments. N ¼ 6. *Po0.05, **Po0.01 vs. untreated control (0 h group).

Article Snippet: In addition, mice were intraperitoneally administered neutralizing goat anti-IL-1b polyclonal antibody or normal goat IgG (both from R&D Systems) at a dose of 100 mg per mouse at 0 and 6 h after indomethacin treatment.

Techniques: Activation Assay, Expressing, Western Blot, Control

Journal: Mucosal immunology

Article Title: Mechanisms of NLRP3 inflammasome activation and its role in NSAID-induced enteropathy.

doi: 10.1038/mi.2015.89

Figure Lengend Snippet: Figure 3 The role of interleukin-1b (IL-1b) in the development of indomethacin-induced small intestinal damage. (a) Mice were intraperitoneally injected with mouse recombinant IL-1b at doses of 0.01–1 mg kg 1 or vehicle 3 h after indomethacin treatment, whereas some mice were given recombinant IL-1b alone. N ¼ 5–7. (b) Mice were intraperitoneally administered neutralizing goat anti-IL-1b polyclonal antibody or vehicle at a dose of 100 mg per mouse 0 and 6 h after indomethacin treatment. N ¼ 6. *Po0.05 vs. vehicle-treated group. (c, d) Representative images of macroscopically visible lesions stained with Evans blue and histological staining 24 h after indomethacin administration in mice given recombinant IL-1b (c) or neutralizing anti-IL-1b antibody (d). Data in a–d are representative of three independent experiments.

Article Snippet: In addition, mice were intraperitoneally administered neutralizing goat anti-IL-1b polyclonal antibody or normal goat IgG (both from R&D Systems) at a dose of 100 mg per mouse at 0 and 6 h after indomethacin treatment.

Techniques: Injection, Recombinant, Staining

Journal: Mucosal immunology

Article Title: Mechanisms of NLRP3 inflammasome activation and its role in NSAID-induced enteropathy.

doi: 10.1038/mi.2015.89

Figure Lengend Snippet: Figure 5 The roles of NLRP3 and caspase-1 (Casp-1) in the activation of interleukin-1b (IL-1b) in indomethacin-induced small intestinal damage. Wild-type (WT), NLRP3 / , and caspase-1 / (Casp-1 / ) mice were administered 10 mg kg 1 indomethacin by gavage and killed 24 h later. (a, b) IL-1b expression level was determined by quantitative reverse transcription–PCR, whereas the concentration of IL-1b was measured by enzyme- linked immunosorbent assay. N ¼ 5–9. (c) Representative western blots for IL-1b and Casp-1. (d–g) Expression levels of (d) pro-IL-1b, (e) mature IL-1b, (f) pro-Casp-1, and (g) cleaved Casp-1 normalized to b-actin. Data are from a single experiment, representative of at least two independent experiments. N ¼ 4–9. *Po0.05, **Po0.01 vs. untreated control; #P o0.05, ##P o0.01 vs. WT mice. (h) The effect of exogenous IL-1b on indomethacin-induced damage in NLRP3 / and Casp-1 / mice. WT, NLRP3 / , and Casp-1 / mice were given intraperitoneal injection of mouse recombinant IL-1b at a dose of 0.1 mg kg 1 3 h after indomethacin treatment. N ¼ 5–7. **Po 0.01 vs. WT mice. Data in a–g are representative of at least three experiments; data in h are representative of two experiments.

Article Snippet: In addition, mice were intraperitoneally administered neutralizing goat anti-IL-1b polyclonal antibody or normal goat IgG (both from R&D Systems) at a dose of 100 mg per mouse at 0 and 6 h after indomethacin treatment.

Techniques: Activation Assay, Expressing, Reverse Transcription, Concentration Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Control, Injection, Recombinant

Journal: Mucosal immunology

Article Title: Mechanisms of NLRP3 inflammasome activation and its role in NSAID-induced enteropathy.

doi: 10.1038/mi.2015.89

Figure Lengend Snippet: Figure 6 Localization of interleukin-1b (IL-1b), NLRP3,and cleaved caspase-1 indamaged small intestinal mucosa. Expression of (a)IL-1b,(b) NLRP3, and (c) cleaved caspase-1 and their colocalization with F4/80 (a marker of mature macrophages) were determined using an immunofluorescence method. Immunofluorescent images are representative of three independent experiments.

Article Snippet: In addition, mice were intraperitoneally administered neutralizing goat anti-IL-1b polyclonal antibody or normal goat IgG (both from R&D Systems) at a dose of 100 mg per mouse at 0 and 6 h after indomethacin treatment.

Techniques: Expressing, Marker, Immunofluorescence

Journal: Mucosal immunology

Article Title: Mechanisms of NLRP3 inflammasome activation and its role in NSAID-induced enteropathy.

doi: 10.1038/mi.2015.89

Figure Lengend Snippet: Figure 7 The roles of macrophages in indomethacin-induced small intestinal damage and activation of interleukin-1b (IL-1b). Mice with or without macrophage depletion by clodronate-encapsulated liposomes were administered 10 mg kg 1 indomethacin by gavage. Some mice were killed 6 h after treatment, and the expression levels of IL-1b were measured. The remaining mice were killed 24 h later to measure the lesion index. (a) The effect of macrophage depletion on indomethacin-induced small intestinal damage. N ¼ 6. **Po0.01 vs. vehicle-treated mice. (b) Representative IL-1b western blot. (c, d) Expression levels of pro-IL-1b and mature IL-1b normalized to b-actin. Data are from a single experiment, representative of three independent experiments. N ¼ 5–6. *Po0.05, **Po0.01 vs. vehicle-treated control. Data in a are representative of three experiments; data in b–d are representative of two experiments.

Article Snippet: In addition, mice were intraperitoneally administered neutralizing goat anti-IL-1b polyclonal antibody or normal goat IgG (both from R&D Systems) at a dose of 100 mg per mouse at 0 and 6 h after indomethacin treatment.

Techniques: Activation Assay, Liposomes, Expressing, Western Blot, Control

Journal: Mucosal immunology

Article Title: Mechanisms of NLRP3 inflammasome activation and its role in NSAID-induced enteropathy.

doi: 10.1038/mi.2015.89

Figure Lengend Snippet: Figure 8 The roles of P2X7 receptor in the activation of the NLRP3 inflammasome during the development of nonsteroidal anti-inflammatory drug (NSAID)-induced enteropathy. Mice were intraperitoneally given apyrase (APY) or Brilliant Blue G (BBG) after indomethacin treatment. Some of the mice were killed 6 h later and the expression levels of interleukin-1b (IL-1b) and components of the NLRP3 inflammasome were measured. The remaining mice were killed 24 h later to measure the lesion index. (a) Representative western blots for IL-1b and caspase-1 (Casp-1). (b–e) Expression levels of (b) pro- IL-1b, (c) mature IL-1b, (d) pro-Casp-1, and (e) cleaved Casp-1 normalized to b-actin. Data are from a single experiment, representative of three independent experiments. N ¼ 4–7. *Po0.05, **Po0.01 vs. untreated control, #Po0.05 vs. vehicle-administered mice with indomethacin treatment. (f) The role of P2X7 receptor in the expression of NLRP3 and IL-1b. N ¼ 7–8. **Po0.01 vs. untreated control. (g) The effect of blocking P2X7 signaling on indomethacin-induced small intestinal damage. N ¼ 6. **Po0.01 vs. vehicle-treated control. (h) Localization of P2X7 receptors in damaged small intestinal mucosa. Data in a–f and h are representative of three experiments; data in g are representative of two experiments.

Article Snippet: In addition, mice were intraperitoneally administered neutralizing goat anti-IL-1b polyclonal antibody or normal goat IgG (both from R&D Systems) at a dose of 100 mg per mouse at 0 and 6 h after indomethacin treatment.

Techniques: Activation Assay, Expressing, Western Blot, Control, Blocking Assay

Journal: Scandinavian journal of immunology

Article Title: Morphological characterization of receptor activator of NFkappaB ligand (RANKL) and IL-1beta expression in rodent collagen-induced arthritis.

doi: 10.1111/j.1365-3083.2005.01632.x

Figure Lengend Snippet: Figure 3 Representative micrographs illustrat- ing brown (DAB) immunoperoxidase staining of synovial tissue from an arthritic ankle (day 21 post immunization). Sequential sections were stained for expression of interleukin 1b (IL-1b) (A), receptor activator of nuclear factor- kB ligand (RANKL) (B), Major histocompat- ibility complex II (C), ED1, a marker for rat monocytes/macrophages (D), a/b-T cell recep- tor (R73) (E), and an irrelevant isotype- matched control (F). Note the abundant expression of IL-1b superficially in the articular cartilage as compared to the paucity of RANKL expressing chondrocytes. Original magnifica- tion 125.

Article Snippet: The primary antibodies used were a goat anti-TRANCE/ TNFSF11 antibody (AF462, R & D Systems, Minneapolis, MN, USA) and a goat anti-IL-1b antibody (AF-501-NA, R & D Systems).

Techniques: Immunoperoxidase Staining, Staining, Expressing, Marker, Control

Journal: Scandinavian journal of immunology

Article Title: Morphological characterization of receptor activator of NFkappaB ligand (RANKL) and IL-1beta expression in rodent collagen-induced arthritis.

doi: 10.1111/j.1365-3083.2005.01632.x

Figure Lengend Snippet: Figure2 Quantified expression of interleukin 1b (IL-1b) and receptor acti- vator of nuclear factor-kB ligand (RANKL) during the course of collagen- induced arthritis. The cytokine expression were studied and quantified at a magnification of 250 using a computerized image analysis system. Each bar represent an individual animal and express the total positive area for the given cytokine in the synovial tissue within three microscopic fields (0.7mm) above and below three joints of the hindpaw, as indicated in Material and Methods.

Article Snippet: The primary antibodies used were a goat anti-TRANCE/ TNFSF11 antibody (AF462, R & D Systems, Minneapolis, MN, USA) and a goat anti-IL-1b antibody (AF-501-NA, R & D Systems).

Techniques: Expressing

Journal: Scandinavian journal of immunology

Article Title: Morphological characterization of receptor activator of NFkappaB ligand (RANKL) and IL-1beta expression in rodent collagen-induced arthritis.

doi: 10.1111/j.1365-3083.2005.01632.x

Figure Lengend Snippet: Figure 4 Representative micrographs illustrating brown (DAB) immuno- peroxidase staining or purple tartrate-resistant acid phosphatase (TRAP) staining of an area of typical subchondral bone erosion at high magnifica- tion in an arthritic ankle (day 28 post immunization). A co-localization of interleukin-1b(IL-1b) (A), receptor activator of nuclearfactor-kB ligand (RANKL) (B), and TRAPþ-expressing cells (C) is demonstrated. Original magnification 500.

Article Snippet: The primary antibodies used were a goat anti-TRANCE/ TNFSF11 antibody (AF462, R & D Systems, Minneapolis, MN, USA) and a goat anti-IL-1b antibody (AF-501-NA, R & D Systems).

Techniques: Staining, Expressing