Journal: Cell Death & Disease

Article Title: P2Y 14 receptor has a critical role in acute gouty arthritis by regulating pyroptosis of macrophages

doi: 10.1038/s41419-020-2609-7

Figure Lengend Snippet: a The acute gouty arthritis was induced by intra-articular injection with MSU solution. P2Y 14 R-KO rats exhibited improved joints swelling caused by MSU. b The circumference of rat ankle joint was increased after the injection of MSU in WT rats but not P2Y 14 R-KO rats. The injected ankle joint circumference of each rat was determined at 0, 2, 4, 8, 12, 24 h after MSU stimulation ( n = 6). c Histopathologic evaluation exhibited a remarkable elevation in the inflammatory infiltration of synovial tissue in WT but not P2Y 14 R-KO rats after MSU exposure. d The macrophages derived from P2Y 14 R-KO synovium exhibited a lower positivity of active Caspase-1 and PI double staining in pyroptosis assay ( n = 4). e After transfection for 48 h, the THP-1 cells were stimulated by MSU. The rate of pyroptotic cell death was examined with active PI and Caspase-1 double staining by flow cytometry ( n = 4). P2Y 14 R knockdown led to a decreased rate of pyroptotic cell death. The data were presented as means ± SDs. One-way analysis of variance (ANOVA) with Tukey multiple comparison test was performed. Compared with WT/NC + vehicle group: # P < 0.05, ## P < 0.01, ### P < 0.001. Compared with WT/NC + MSU group: * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: NLRP3 (bs-10021R), ASC (bs-6741R), caspase-1 (bs-10442R), and β-actin (bs-0061R) antibodies for western blot were obtained from Bioss (Beijing, China).

Techniques: Injection, Derivative Assay, Double Staining, Transfection, Flow Cytometry

Journal: Cell Death & Disease

Article Title: P2Y 14 receptor has a critical role in acute gouty arthritis by regulating pyroptosis of macrophages

doi: 10.1038/s41419-020-2609-7

Figure Lengend Snippet: As a potent adenylate cyclase (AC) activator, Forskolin is used to increase the intracellular level of the second messenger cAMP. Intra-articular administration of Forskolin was given to WT rats prior to MSU induction. a The intracellular cAMP level in synovial tissue increased significantly after Forskolin treatment ( n = 6). b cAMP elevation induced by Forskolin prevented the joint swelling. Representative photographs to show the swelling of joints are presented. c Forskolin-induced cAMP elevation alleviated the injected ankle joint circumference under MSU challenge ( n = 6). d Forskolin treatment efficiently inhibited inflammatory cell infiltration when compared MSU group in histopathologic test. e The macrophages derived from synovium of Forskolin-treated rats exhibited a lower positivity of active Caspase-1 and PI double staining in pyroptosis assay by flow cytometry ( n = 4). f Forskolin treatment inhibited the colocalization intensity of NLRP3 and ASC in MSU-stimulated WT animals in immunofluorescence staining. NLRP3 protein was marked with Alexa Fluor 488 (Green). ASC protein was marked with Alexa Fluor 647 (Red). DAPI (Blue) was used to mark the nucleus. g Western blotting showed Forskolin treatment inhibited MSU-induced NLRP3 inflammasome activation in WT rats. The relative optical density was exhibited in the ( n = 4). The data were presented as means ± SDs. Compared with WT + vehicle group: # P < 0.05, ## P < 0.01, ### P < 0.001. Compared with WT + MSU group: * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: NLRP3 (bs-10021R), ASC (bs-6741R), caspase-1 (bs-10442R), and β-actin (bs-0061R) antibodies for western blot were obtained from Bioss (Beijing, China).

Techniques: Injection, Derivative Assay, Double Staining, Flow Cytometry, Immunofluorescence, Staining, Western Blot, Activation Assay

Journal: Cell Death & Disease

Article Title: P2Y 14 receptor has a critical role in acute gouty arthritis by regulating pyroptosis of macrophages

doi: 10.1038/s41419-020-2609-7

Figure Lengend Snippet: 15 min pre-treatment of Forskolin was exposed to THP-1 cells followed by MSU model for 12 h. a The intracellular cAMP level in THP-1 cells was verified to rise after Forskolin treatment by cAMP assay kit ( n = 4). b Forskolin-induced cAMP elevation suppressed the release of IL-1β in THP-1 cells ( n = 4). c The THP-1 cells treated with Forskolin presented a decreased rate of pyroptotic cell death in active Caspase-1 and PI double staining by flow cytometry ( n = 4). d Western blotting showed that the enhanced cAMP level attenuated the MSU-induced activation of the NLRP3 signaling pathway in THP-1 cells treated with Forskolin. The relative optical density was exhibited in the ( n = 4). e Immunofluorescence assay confirmed that enhanced cAMP level attenuated MSU-induced NLRP3 inflammasome activation, as evidenced by decreased expression and colocalization of NLRP3 and ASC in THP-1 cells treated with Forskolin. NLRP3 protein was marked with Alexa Fluor 488 (Green). ASC protein was marked with Alexa Fluor 647 (Red). DAPI (Blue) was used to mark the nucleus. The data were presented as means ± SDs. One-way analysis of variance (ANOVA) with Tukey multiple comparison test was performed. Compared with vehicle group: # P < 0.05, ## P < 0.01, ### P < 0.001. Compared with MSU group: * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: NLRP3 (bs-10021R), ASC (bs-6741R), caspase-1 (bs-10442R), and β-actin (bs-0061R) antibodies for western blot were obtained from Bioss (Beijing, China).

Techniques: cAMP Assay, Double Staining, Flow Cytometry, Western Blot, Activation Assay, Immunofluorescence, Expressing

Journal: Cell Death & Disease

Article Title: P2Y 14 receptor has a critical role in acute gouty arthritis by regulating pyroptosis of macrophages

doi: 10.1038/s41419-020-2609-7

Figure Lengend Snippet: P2Y 14 R siRNA was used to transfect THP-1 cells with Lipofectamine 2000 for 48 h. 15 min pre-treatment of SQ22536 was exposed to THP-1 cells followed by MSU model for 12 h. a The intracellular cAMP level in THP-1 cells was verified to reduce by cAMP assay kit in siP2Y 14 R THP-1 cells after SQ22536 treatment ( n = 4). b SQ22536-induced cAMP reduction promoted the release of IL-1β in siP2Y 14 R THP-1 cells ( n = 4). c Decreased cAMP in THP-1 cells restored the siP2Y 14 R-derived protective effect on MSU-stimulated pyroptosis with a rising rate of active Caspase-1 and PI double positivity by flow cytometry ( n = 4). d Western blotting showed that the decreased cAMP level promoted the MSU-induced activation of the NLRP3 signaling pathway in THP-1 cells treated with SQ22536 under P2Y 14 R knockdown. The relative optical density was exhibited in the ( n = 4). e Immunofluorescence assay confirmed that decreased cAMP recovered NLRP3 inflammasome activation in THP-1 treated with P2Y 14 R knockdown, as evidenced by increased expression and colocalization of NLRP3 and ASC after SQ22536 administration. NLRP3 protein was marked with Alexa Fluor 488 (Green). ASC protein was marked with Alexa Fluor 647 (Red). DAPI (Blue) was used to mark the nucleus. The data were presented as means ± SDs. One-way analysis of variance (ANOVA) with Tukey multiple comparison test was performed. Compared with siP2Y 14 R + vehicle group: # P < 0.05, ## P < 0.01, ### P < 0.001. Compared with siP2Y 14 R + MSU group: * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: NLRP3 (bs-10021R), ASC (bs-6741R), caspase-1 (bs-10442R), and β-actin (bs-0061R) antibodies for western blot were obtained from Bioss (Beijing, China).

Techniques: cAMP Assay, Derivative Assay, Flow Cytometry, Western Blot, Activation Assay, Immunofluorescence, Expressing

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