Journal: Immunology

Article Title: NLRP 3 inflammasome mediates interleukin‐1 β production in immune cells in response to Acinetobacter baumannii and contributes to pulmonary inflammation in mice

doi: 10.1111/imm.12704

Figure Lengend Snippet: NLRP3, ASC and caspase‐1 are required for interleukin‐1β (IL‐1β) production in bone‐marrow‐derived macrophages (BMDMs) in response to Acinetobacter baumannii. BMDMs from wild‐type and NLRC4‐, NLRP3‐, ASC‐ and caspase‐1/11‐deficient mice were infected with A. baumannii at the indicated multiplicities of infection (MOIs) (a–c and f–h). For an inhibitor assay, BMDMs (d) and murine bronchoepithelial MH‐S cells (e) were pretreated with various doses of glyburide (an NLRP3 inhibitor) for 2 hr and subsequently infected with A. baumannii at an MOI of 10. One hour after infection, the cells were treated with gentamicin to inhibit extracellular bacterial growth and further incubated for 24 hr. The concentrations of IL‐1β, IL‐6 and tumour necrosis factor‐α (TNF‐α) in culture supernatants were determined by ELISA. The results are from one experiment that is representative of three independent experiments and are expressed as means ± SD. *P < 0·05, **P < 0·01 and ***P < 0·001.

Article Snippet: The membranes were immunoblotted with the primary antibodies anti‐mouse caspase‐1 (Enzo Life Science, Farmingdale, NY), anti‐mouse‐IL‐1 β (R&D Systems) and anti‐ β‐ actin (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Derivative Assay, Infection, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Immunology

Article Title: NLRP 3 inflammasome mediates interleukin‐1 β production in immune cells in response to Acinetobacter baumannii and contributes to pulmonary inflammation in mice

doi: 10.1111/imm.12704

Figure Lengend Snippet: NLRP3/ASC inflammasomes contribute to caspase‐1 activation and interleukin‐1β (IL‐1β) maturation during Acinetobacter baumannii infection in bone‐marrow‐derived macrophages (BMDMs). BMDMs from wild‐type and NLRC4‐, NLRP3‐, ASC‐ and caspase‐1/11‐deficient mice were infected with A. baumannii at a multiplicity of infection (MOI) of 10 for 24 hr with gentamycin treatment as previously described (a and b). Culture supernatants and cell lysates were separately prepared and used for a Western blot analysis to detect cleaved and immature forms of caspase‐1 and IL‐1β. Antibody to β‐actin was used as a loading control.

Article Snippet: The membranes were immunoblotted with the primary antibodies anti‐mouse caspase‐1 (Enzo Life Science, Farmingdale, NY), anti‐mouse‐IL‐1 β (R&D Systems) and anti‐ β‐ actin (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Activation Assay, Infection, Derivative Assay, Western Blot

Journal: Immunology

Article Title: NLRP 3 inflammasome mediates interleukin‐1 β production in immune cells in response to Acinetobacter baumannii and contributes to pulmonary inflammation in mice

doi: 10.1111/imm.12704

Figure Lengend Snippet: Production of interleukin‐1β (IL‐1β) in bronchoalveolar lavage (BAL) fluid is impaired in NLRP3‐ and caspase‐1/11‐deficient mice infected with Acinetobacter baumannii. Wild‐type and NLRP3‐ or caspase‐1/11‐deficient mice were intranasally infected with A. baumannii (3 × 107 colony‐forming units in PBS) and killed at 6 hr and 1 day after infection. BAL fluid was collected from each mouse, and the levels of IL‐1β (a and b), IL‐6 (c and d), tumour necrosis factor‐α (TNF‐α) (e and f) and IL‐10 (g and h) were measured by ELISA. The results were expressed as means ± SD. *P < 0·05, **P < 0·01 and ***P < 0·001.

Article Snippet: The membranes were immunoblotted with the primary antibodies anti‐mouse caspase‐1 (Enzo Life Science, Farmingdale, NY), anti‐mouse‐IL‐1 β (R&D Systems) and anti‐ β‐ actin (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Infection, Enzyme-linked Immunosorbent Assay

Journal: Immunology

Article Title: NLRP 3 inflammasome mediates interleukin‐1 β production in immune cells in response to Acinetobacter baumannii and contributes to pulmonary inflammation in mice

doi: 10.1111/imm.12704

Figure Lengend Snippet: NLRP3 and caspase‐1 deficiency reduces lung pathology in Acinetobacter baumannii‐infected mice, although it does not affect bacterial clearance. Wild‐type and NLRP3‐ or caspase‐1/11‐deficient mice were intranasally infected with A. baumannii (3 × 107 colony‐forming units in PBS) and killed at 6 hr, 1 day or 3 days after infection. Bacterial loads in bronchoalveolar lavage (BAL) fluid (a and b) or lung homogenates (c and d) were counted by plating assay, and lung histopathology was evaluated (e; × 100 magnification) as described in the Materials and methods. Histological scores are shown as means ± SD (f). These results were from one experiment that is representative of two independent experiments. *P < 0·05 and **P < 0·01. [Colour figure can be viewed at wileyonlinelibrary. com]

Article Snippet: The membranes were immunoblotted with the primary antibodies anti‐mouse caspase‐1 (Enzo Life Science, Farmingdale, NY), anti‐mouse‐IL‐1 β (R&D Systems) and anti‐ β‐ actin (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Infection, Histopathology

Journal: Immunology

Article Title: NLRP 3 inflammasome mediates interleukin‐1 β production in immune cells in response to Acinetobacter baumannii and contributes to pulmonary inflammation in mice

doi: 10.1111/imm.12704

Figure Lengend Snippet: NLRP3 and caspase‐1 are involved in interleukin‐1β (IL‐1β) production in neutrophils in response to Acinetobacter baumannii. Thioglycollate‐elicited neutrophils (Nphs), lung epithelial cells and L‐929 cells were seeded in a 48‐well plate (2 × 105 cells/well) and infected with A. baumannii at a multiplicity of infection (MOI) of 1/10 for 24 hr (a). In addition, neutrophils from wild‐type, NLRP3‐ and caspase‐1/11‐deficient mice were infected with different doses of A. baumannii for 24 hr (b–d). Levels of IL‐1β, IL‐6 and tumour necrosis factor‐α (TNF‐α) in culture supernatants were measured by ELISA. The results were expressed as means ± SD. ***P < 0·001.

Article Snippet: The membranes were immunoblotted with the primary antibodies anti‐mouse caspase‐1 (Enzo Life Science, Farmingdale, NY), anti‐mouse‐IL‐1 β (R&D Systems) and anti‐ β‐ actin (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Infection, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Serum Amyloid A Induces NLRP-3-Mediated IL-1β Secretion in Neutrophils

doi: 10.1371/journal.pone.0096703

Figure Lengend Snippet: A Neutrophils were stimulated with SAA (5 µg/ml) in the presence or absence of Z-YVAD-FMK for 8 h. After stimulation, supernatants were analyzed for IL-1β production using ELISA. Values represent the mean ± SD of two independent experiments. * p <0.001 compared to SAA-stimulated neutrophils. B Neutrophils were stimulated with SAA (5 µg/ml) in the presence or absence of Z-YVAD-FMK for 8 h. Supernatants were analyzed by western blot analysis for the presence of mature IL-1β. C Neutrophils were stimulated with SAA (5 µg/ml) in the presence or absence of Z-YVAD-FMK for 8 h. Culture supernatants (SN) and cellular lysates (CL) were analyzed by immunoblot using anti-caspase-1 Ab. Caspase-1 (p20, cleaved subunit; p45, precursor). Two experiments were performed using different neutrophils and a representative result is shown.

Article Snippet: Anti-phosphotyrosine (clone 4G10), anti-Syk (clone4D10) and anti-caspase-1 antibodies were purchased from MERCK MILLIPORE (Billerica,MA USA).

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot

Journal: PLoS ONE

Article Title: Serum Amyloid A Induces NLRP-3-Mediated IL-1β Secretion in Neutrophils

doi: 10.1371/journal.pone.0096703

Figure Lengend Snippet: A Neutrophils were pretreated or untreated with the indicated concentrations of R406 for 1(5 µg/ml) for 8 hr. After stimulation, supernatants were analyzed by western blot analysis for the presence of mature IL-1β. Three experiments were performed using different neutrophils and a representative result is shown. B Neutrophils were pretreated or untreated with the indicated concentrations of R406 for 1 hr. The cells were stimulated with SAA (5 µg/ml) for 8 hr. After stimulation, culture supernatants (SN) and cellular lysates (CL) were analyzed by immunoblot using anti-caspase-1 Ab. Caspase-1 (p20, cleaved subunit; p45, precursor). Three experiments were performed using different neutrophils and a representative result is shown.

Article Snippet: Anti-phosphotyrosine (clone 4G10), anti-Syk (clone4D10) and anti-caspase-1 antibodies were purchased from MERCK MILLIPORE (Billerica,MA USA).

Techniques: Western Blot