msu Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Millipore msu preparation msu
    Cross-presentation of NLRs and TLRs in LM-treated DCs DCs were harvested after 24 hours of LM infection (at a MOI of 10) (or of incubation without LM) and then treated to silence or stimulate NLRP3 expression with corresponding receptor agonists (LPS, TLR4 agonist; <t>MDP,</t> NOD1/2 agonist; <t>MSU,</t> NLRP3 agonist) for 6-8 h as described in the Materials and Methods section. Expression of the functional DC molecules CD80 and CD86 was assessed by flow cytometry, and the peak with gray filled represents the control (A and B). Supernatants were collected for quantification of IL-12p70, IL-β, TNF-αand IFN-γ by ELISA (C). Protein levels of NOD1, NLRP3 and TLR4 (D) and expression of NF-kB and caspase-1 (E) were detected by western blot. GAPDH was used as the internal control, and summary statistics are depicted in the histograms. Each data point represents the mean ± SEM from three independent experiments (*p
    Msu Preparation Msu, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/msu preparation msu/product/Millipore
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    msu preparation msu - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

    91
    InvivoGen msu
    Suppression of RIPK3 and MLKL prevents crystal cytotoxicity. ( a – c ) Mouse tubular epithelial cells were transfected with specific small inhibitor (si) RNA for RIPK3 and MLKL or a control siRNA of scrambled sequence before being exposed to crystals of <t>CaOx</t> (1,000 μg ml −1 ), <t>MSU</t> (500 μg ml −1 ), CPPD (500 μg ml −1 ) and cystine (500 μg ml −1 ). Cell viability was assessed by MTT assay (a) and cell death was assessed quantifying PI positivity (b) and C shows representative images 24 h later. Original image magnification: × 200, scale bar, 100 μm. ( d ) Mouse tubular epithelial cells were pretreated with RIPK3 inhibitor dabrafenib before exposing to different type crystals. Cell viability was assessed by MTT assay 24 h later. Data are expressed as mean±s.e.m. of three independent experiments, and was analysed using Student's t -test. Baseline viability is set as 100%. * P
    Msu, supplied by InvivoGen, used in various techniques. Bioz Stars score: 91/100, based on 148 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/msu/product/InvivoGen
    Average 91 stars, based on 148 article reviews
    Price from $9.99 to $1999.99
    msu - by Bioz Stars, 2020-08
    91/100 stars
      Buy from Supplier

    97
    InvivoGen msu crystals
    MicroRNA (miR, miRNA)-488 and miR-920 suppress monosodium urate <t>(MSU)-induced</t> expression of proinflammatory <t>cytokines</t> in THP-1 cells. The miRNA mimics or negative control (NC) mimics (50 nM) were transfected into THP-1 cells using Lipofectamine RNAiMAX reagent in accordance with the manufacturer’s instructions. After 24 h of transfection, cells were stimulated for 3 h with 0.5 μM 12-myristate 13-acetate. Then, cells were washed and stimulated with 250 μg/ml MSU crystals for 24 h to detect the production of proinflammatory cytokines. After the treatment, the cells were collected and analyzed by quantitative real-time polymerase chain reaction ( a , c ). The cell culture supernatants were also collected to detect the concentrations of interleukin (IL)-8 and tumor necrosis factor (TNF)-α by enzyme-linked immunosorbent assay ( b , d ). Values are expressed as mean ± SEM of three independent experiments, each of which was run in triplicate. # P
    Msu Crystals, supplied by InvivoGen, used in various techniques. Bioz Stars score: 97/100, based on 129 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/msu crystals/product/InvivoGen
    Average 97 stars, based on 129 article reviews
    Price from $9.99 to $1999.99
    msu crystals - by Bioz Stars, 2020-08
    97/100 stars
      Buy from Supplier

    91
    InvivoGen monosodium urate msu crystals
    iNKT Cells Induce IL-1β Secretion by <t>BMDCs</t> in a CD1d-Dependent Manner (A and B) BMDCs were left unprimed or primed with Pam3CSK4 (P3C; 0.5 μg/mL) for 24 h and subsequently co-cultured with iNKT cells for 24 h in the (A) absence or (B) presence of αGalCer (50 ng/mL). IL-1β release was quantified by ELISA. As a control, BMDCs were primed with P3C for 4 h, followed by 6-h stimulation with <t>MSU</t> (100 μg/mL). The same concentrations of P3C, αGalCer, and MSU were used throughout all experiments. (C and D) P3C-primed BMDCs were cultured alone or with iNKT cells for the indicated times in the presence or absence of αGalCer. IL-1β release was quantified by ELISA (C), and cell-associated and extracellular IL-1β were analyzed by immunoblot (D). As a control, BMDCs were primed with P3C for 4 h, followed by 1-h stimulation with nigericin (10 μM). (E) P3C-primed BMDCs from WT and CD1d −/− mice were co-cultured with iNKT cells for the indicated times in the absence or presence of αGalCer. IL-1β release was quantified by ELISA. P3C-primed BMDCs stimulated with nigericin were used as a control. Error bars represent mean + SD of triplicate samples. Each panel is representative of at least three different experiments. *p
    Monosodium Urate Msu Crystals, supplied by InvivoGen, used in various techniques. Bioz Stars score: 91/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/monosodium urate msu crystals/product/InvivoGen
    Average 91 stars, based on 24 article reviews
    Price from $9.99 to $1999.99
    monosodium urate msu crystals - by Bioz Stars, 2020-08
    91/100 stars
      Buy from Supplier

    88
    InvivoGen msu tlrl msu
    iNKT Cells Induce IL-1β Secretion by <t>BMDCs</t> in a CD1d-Dependent Manner (A and B) BMDCs were left unprimed or primed with Pam3CSK4 (P3C; 0.5 μg/mL) for 24 h and subsequently co-cultured with iNKT cells for 24 h in the (A) absence or (B) presence of αGalCer (50 ng/mL). IL-1β release was quantified by ELISA. As a control, BMDCs were primed with P3C for 4 h, followed by 6-h stimulation with <t>MSU</t> (100 μg/mL). The same concentrations of P3C, αGalCer, and MSU were used throughout all experiments. (C and D) P3C-primed BMDCs were cultured alone or with iNKT cells for the indicated times in the presence or absence of αGalCer. IL-1β release was quantified by ELISA (C), and cell-associated and extracellular IL-1β were analyzed by immunoblot (D). As a control, BMDCs were primed with P3C for 4 h, followed by 1-h stimulation with nigericin (10 μM). (E) P3C-primed BMDCs from WT and CD1d −/− mice were co-cultured with iNKT cells for the indicated times in the absence or presence of αGalCer. IL-1β release was quantified by ELISA. P3C-primed BMDCs stimulated with nigericin were used as a control. Error bars represent mean + SD of triplicate samples. Each panel is representative of at least three different experiments. *p
    Msu Tlrl Msu, supplied by InvivoGen, used in various techniques. Bioz Stars score: 88/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/msu tlrl msu/product/InvivoGen
    Average 88 stars, based on 17 article reviews
    Price from $9.99 to $1999.99
    msu tlrl msu - by Bioz Stars, 2020-08
    88/100 stars
      Buy from Supplier

    91
    Enzo Biochem msu crystals
    Gasdermin D pore formation and pyroptosis are not involved in EEA1 release. ( A ) Immunoblot analysis of EEA1 and IL-1β on cell lysate and cell-free supernatant from LPS-primed BMDMs from wild type or Casp1/11 −/− after stimulation with nigericin, ATP, E . coli or <t>MSU</t> crystals as indicated. IL-1β released to the extracellular medium was detected by ELISA (bottom). ( B ) LDH release from macrophages treated as in A. ( C ) Immunoblot analysis of EEA1 on cell lysate and cell-free supernatant from BMDMs wild-type and Gsdmd −/− after nigericin stimulation. ( D ) Immunoblot analysis of <t>caspase-1</t> and IL-1β on cell lysate and cell-free supernatant from wild-type or Gsdmd −/− BMDMs after nigericin stimulation in the presence or absence of the caspase-1 inhibitor Ac-YVAD. ( E , F ) ELISA for released IL-1β ( E ) or LDH ( F ) from macrophages treated as in D. ( G ) Immunoblot analysis of ASC and NLRP3 on cell-free supernatant from macrophages stimulated as in D. ( H ) Yo-Pro uptake in macrophages treated as in D. Blots are representative of three to four independent experiments. Nitrocellulose membranes were cropped after protein blotting and then inmunodetection was carried out separately for each antibody. Complete blots for IL-1β and Caspase-1 are showed as supplementary. Data are presented as average ± sem from n = 3 independent experiments.
    Msu Crystals, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 91/100, based on 72 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/msu crystals/product/Enzo Biochem
    Average 91 stars, based on 72 article reviews
    Price from $9.99 to $1999.99
    msu crystals - by Bioz Stars, 2020-08
    91/100 stars
      Buy from Supplier

    91
    Thermo Fisher msu crystals
    Gasdermin D pore formation and pyroptosis are not involved in EEA1 release. ( A ) Immunoblot analysis of EEA1 and IL-1β on cell lysate and cell-free supernatant from LPS-primed BMDMs from wild type or Casp1/11 −/− after stimulation with nigericin, ATP, E . coli or <t>MSU</t> crystals as indicated. IL-1β released to the extracellular medium was detected by ELISA (bottom). ( B ) LDH release from macrophages treated as in A. ( C ) Immunoblot analysis of EEA1 on cell lysate and cell-free supernatant from BMDMs wild-type and Gsdmd −/− after nigericin stimulation. ( D ) Immunoblot analysis of <t>caspase-1</t> and IL-1β on cell lysate and cell-free supernatant from wild-type or Gsdmd −/− BMDMs after nigericin stimulation in the presence or absence of the caspase-1 inhibitor Ac-YVAD. ( E , F ) ELISA for released IL-1β ( E ) or LDH ( F ) from macrophages treated as in D. ( G ) Immunoblot analysis of ASC and NLRP3 on cell-free supernatant from macrophages stimulated as in D. ( H ) Yo-Pro uptake in macrophages treated as in D. Blots are representative of three to four independent experiments. Nitrocellulose membranes were cropped after protein blotting and then inmunodetection was carried out separately for each antibody. Complete blots for IL-1β and Caspase-1 are showed as supplementary. Data are presented as average ± sem from n = 3 independent experiments.
    Msu Crystals, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 96 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/msu crystals/product/Thermo Fisher
    Average 91 stars, based on 96 article reviews
    Price from $9.99 to $1999.99
    msu crystals - by Bioz Stars, 2020-08
    91/100 stars
      Buy from Supplier

    89
    Crystal IS msu crystal
    Four axial spondyloarthritis (AxSpA) patients with monosodium urate <t>(MSU)</t> crystal and radiographic structural damage at the sacroiliac joint. For each set of images, panel a shows the sacroiliac joint on plain radiographs, panel b shows the three-dimensional reconstruction dual-energy computed tomography <t>(DECT)</t> images, panel c shows the corresponding coronal (patient 2 and 4) or axial (patient 1 and 3) DECT images, and panel d shows the corresponding level of computed tomography (CT) images. A large quantity of MSU crystal deposition shown as green was found at the sacroiliac joint or the surrounding area in the DECT images. Four male patients (patient 1–4), aged 36, 44, 23, and 27 years old, had serum uric acid levels of 407 μmol/L, 370 μmol/L, 572 μmol/L, and 464 μmol/L, respectively. They were graded with a scale of 0, 0, II, III at the left sacroiliac joint and 0, I, II, III at the right sacroiliac joint, respectively, on plain radiographs
    Msu Crystal, supplied by Crystal IS, used in various techniques. Bioz Stars score: 89/100, based on 65 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/msu crystal/product/Crystal IS
    Average 89 stars, based on 65 article reviews
    Price from $9.99 to $1999.99
    msu crystal - by Bioz Stars, 2020-08
    89/100 stars
      Buy from Supplier

    91
    FUJIFILM msu crystals
    Cytokine induction in early infection does not involve an autocrine mechanism. (A) After incubation with the indicated amount of recombinant human IL-1Ra for 30 min, miCtrl cells were stimulated with recombinant human IL-1β for 60 min, and the IL-8 secretion was analyzed by ELISA. (B) After incubation with 100 ng/ml IL-1Ra for 30 min, miCtrl cells were infected with S . aureus at an MOI of 4 for the times indicated. IL-8 and TNF-α secretion from each cell line were analyzed by ELISA. (C) After incubation with 20 μM Ac-YVAD-CMK for 30 min, Dox-untreated miCtrl and miNLRP3 cells were infected with S . aureus for 100 min. TNF-α and IL-1β secretion were analyzed by ELISA. (D) THP-1 cells were incubated with 5 μg/ml <t>CHX</t> for 30 min and then infected with S . aureus at an MOI of 4 for the indicated times. TNF-α and IL-1β secretion were analyzed by ELISA. (E) THP-1 cells were treated with CHX and infected as in (D). The total RNA was analyzed for TNF-α and IL-1β mRNA by RT-PCR. (F) ELISA analysis of TNF-α and IL-1β released from THP-1 cells treated with <t>MSU</t> in the presence or absence of 0.63, 1.25, 2.5, 5, and 10 μM Ac-YVAD-CMK for 180 min. (G) ELISA analysis of IL-18 released from THP-1 cells treated as in (C) (left panel) or treated with MSU in the presence or absence of 10 μM of Ac-YVAD-CMK for 20 h (right panel). All results are representative of three independent experiments. ND, not detected.
    Msu Crystals, supplied by FUJIFILM, used in various techniques. Bioz Stars score: 91/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/msu crystals/product/FUJIFILM
    Average 91 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    msu crystals - by Bioz Stars, 2020-08
    91/100 stars
      Buy from Supplier

    91
    Caltag-Medsystems msu crystals
    <t>IL-1β</t> secretion by PBMCs following exposure to <t>MSU</t> crystals or SNPs. Secretion of IL-1β by PBMCs with (main figure) or without (inset) initial exposure to 10 ng/mL LPS for 3 h followed by exposure to MSU crystals (100 μg/mL) or SNPs (100 μg/mL) for a further 3 h. The supernatant was either collected immediately for analysis (3 h; open bars) or following a further 21 h of cell incubation in fresh (i.e. without added particles or MAMPs) TCM (3 + 21 h; black bars). All data are expressed as mean ± SEM ( n = 4). Results were analysed by paired T test in comparison to baseline (B), i.e. non-particle-exposed cells; * p
    Msu Crystals, supplied by Caltag-Medsystems, used in various techniques. Bioz Stars score: 91/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/msu crystals/product/Caltag-Medsystems
    Average 91 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    msu crystals - by Bioz Stars, 2020-08
    91/100 stars
      Buy from Supplier

    91
    Enzo Biochem msu
    Extracellular succinate signals via GPR91 to stimulate macrophages to release <t>IL-1β.</t> (A) GPR91 mRNA expression in WT (Janvier C57BL/6J) inflammatory BMDMs (M-CSF + IFN-γ) ± 100 ng/ml LPS, 500 µM succinate, or 10 ng/ml IL-1β for 24 h. n = 3 of Ct values. Succinate (Succ), IL-1β, and LPS related to basal (=1). Data are representative of three experiments. (B) Succinate levels (mass spectrophotometry area ratio) in medium from cultured BMDMs. Extracellular succinate from WT (littermates; black bars) and Sucnr1 −/− (gray bars), neutral (M, M-CSF), or inflammatory (M + IFN-γ) BMDMs ± 100 ng/ml LPS for 24 h is shown. n = 6 wells. Data are representative of three experiments. (C) IL-1β in supernatants of WT (Janvier C57BL/6J) and Sucnr1 −/− neutral or inflammatory BMDMs ± 100 ng/ml LPS for 24 h. n = 3 wells and are representative of seven experiments. (D) IL-1β mRNA levels from cell lysates from WT (Janvier C57BL/6J) or Sucnr1 −/− inflammatory BMDMs ± 100 ng/ml LPS at 4 h (related to WT basal = 1). n = 2–3 of Ct values. Data are representative of two experiments. (E) IL-1β levels measured in the supernatant of WT (Janvier C57BL/6J) and Sucnr1 −/− inflammatory BMDMs stimulated with 1 ng/ml LPS and 180 µg/ml <t>MSU.</t> n = 5–6 wells. Data are representative of two experiments. (F) Western blot of HIF-1α (representative blot of two experiments) and quantification (two experiments; 100% for no stimulus, WT, and Sucnr1 −/− ) at 6 h after stimulation with 500 µM succinate, 100 ng/ml LPS, or a combination of the two in WT littermate controls and Sucnr1 −/− inflammatory BMDMs. *, P
    Msu, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 91/100, based on 67 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/msu/product/Enzo Biochem
    Average 91 stars, based on 67 article reviews
    Price from $9.99 to $1999.99
    msu - by Bioz Stars, 2020-08
    91/100 stars
      Buy from Supplier

    90
    Merck & Co msu
    Extracellular succinate signals via GPR91 to stimulate macrophages to release <t>IL-1β.</t> (A) GPR91 mRNA expression in WT (Janvier C57BL/6J) inflammatory BMDMs (M-CSF + IFN-γ) ± 100 ng/ml LPS, 500 µM succinate, or 10 ng/ml IL-1β for 24 h. n = 3 of Ct values. Succinate (Succ), IL-1β, and LPS related to basal (=1). Data are representative of three experiments. (B) Succinate levels (mass spectrophotometry area ratio) in medium from cultured BMDMs. Extracellular succinate from WT (littermates; black bars) and Sucnr1 −/− (gray bars), neutral (M, M-CSF), or inflammatory (M + IFN-γ) BMDMs ± 100 ng/ml LPS for 24 h is shown. n = 6 wells. Data are representative of three experiments. (C) IL-1β in supernatants of WT (Janvier C57BL/6J) and Sucnr1 −/− neutral or inflammatory BMDMs ± 100 ng/ml LPS for 24 h. n = 3 wells and are representative of seven experiments. (D) IL-1β mRNA levels from cell lysates from WT (Janvier C57BL/6J) or Sucnr1 −/− inflammatory BMDMs ± 100 ng/ml LPS at 4 h (related to WT basal = 1). n = 2–3 of Ct values. Data are representative of two experiments. (E) IL-1β levels measured in the supernatant of WT (Janvier C57BL/6J) and Sucnr1 −/− inflammatory BMDMs stimulated with 1 ng/ml LPS and 180 µg/ml <t>MSU.</t> n = 5–6 wells. Data are representative of two experiments. (F) Western blot of HIF-1α (representative blot of two experiments) and quantification (two experiments; 100% for no stimulus, WT, and Sucnr1 −/− ) at 6 h after stimulation with 500 µM succinate, 100 ng/ml LPS, or a combination of the two in WT littermate controls and Sucnr1 −/− inflammatory BMDMs. *, P
    Msu, supplied by Merck & Co, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/msu/product/Merck & Co
    Average 90 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    msu - by Bioz Stars, 2020-08
    90/100 stars
      Buy from Supplier

    Image Search Results


    Cross-presentation of NLRs and TLRs in LM-treated DCs DCs were harvested after 24 hours of LM infection (at a MOI of 10) (or of incubation without LM) and then treated to silence or stimulate NLRP3 expression with corresponding receptor agonists (LPS, TLR4 agonist; MDP, NOD1/2 agonist; MSU, NLRP3 agonist) for 6-8 h as described in the Materials and Methods section. Expression of the functional DC molecules CD80 and CD86 was assessed by flow cytometry, and the peak with gray filled represents the control (A and B). Supernatants were collected for quantification of IL-12p70, IL-β, TNF-αand IFN-γ by ELISA (C). Protein levels of NOD1, NLRP3 and TLR4 (D) and expression of NF-kB and caspase-1 (E) were detected by western blot. GAPDH was used as the internal control, and summary statistics are depicted in the histograms. Each data point represents the mean ± SEM from three independent experiments (*p

    Journal: Oncotarget

    Article Title: The attenuated hepatocellular carcinoma-specific Listeria vaccine Lmdd-MPFG prevents tumor occurrence through immune regulation of dendritic cells

    doi:

    Figure Lengend Snippet: Cross-presentation of NLRs and TLRs in LM-treated DCs DCs were harvested after 24 hours of LM infection (at a MOI of 10) (or of incubation without LM) and then treated to silence or stimulate NLRP3 expression with corresponding receptor agonists (LPS, TLR4 agonist; MDP, NOD1/2 agonist; MSU, NLRP3 agonist) for 6-8 h as described in the Materials and Methods section. Expression of the functional DC molecules CD80 and CD86 was assessed by flow cytometry, and the peak with gray filled represents the control (A and B). Supernatants were collected for quantification of IL-12p70, IL-β, TNF-αand IFN-γ by ELISA (C). Protein levels of NOD1, NLRP3 and TLR4 (D) and expression of NF-kB and caspase-1 (E) were detected by western blot. GAPDH was used as the internal control, and summary statistics are depicted in the histograms. Each data point represents the mean ± SEM from three independent experiments (*p

    Article Snippet: BMDCs were stimulated with LPS (1 μg/ml), MDP (10 μg/ml), MSU (250 μg/ml), LPS and MSU together, or MDP and MSU together for 6-8 h. LPS, MDP, MSU were purchased from Sigma Chemical Co. (Sigma, St Louis, MO, USA).

    Techniques: Infection, Incubation, Expressing, Functional Assay, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay, Western Blot

    Suppression of RIPK3 and MLKL prevents crystal cytotoxicity. ( a – c ) Mouse tubular epithelial cells were transfected with specific small inhibitor (si) RNA for RIPK3 and MLKL or a control siRNA of scrambled sequence before being exposed to crystals of CaOx (1,000 μg ml −1 ), MSU (500 μg ml −1 ), CPPD (500 μg ml −1 ) and cystine (500 μg ml −1 ). Cell viability was assessed by MTT assay (a) and cell death was assessed quantifying PI positivity (b) and C shows representative images 24 h later. Original image magnification: × 200, scale bar, 100 μm. ( d ) Mouse tubular epithelial cells were pretreated with RIPK3 inhibitor dabrafenib before exposing to different type crystals. Cell viability was assessed by MTT assay 24 h later. Data are expressed as mean±s.e.m. of three independent experiments, and was analysed using Student's t -test. Baseline viability is set as 100%. * P

    Journal: Nature Communications

    Article Title: Cytotoxicity of crystals involves RIPK3-MLKL-mediated necroptosis

    doi: 10.1038/ncomms10274

    Figure Lengend Snippet: Suppression of RIPK3 and MLKL prevents crystal cytotoxicity. ( a – c ) Mouse tubular epithelial cells were transfected with specific small inhibitor (si) RNA for RIPK3 and MLKL or a control siRNA of scrambled sequence before being exposed to crystals of CaOx (1,000 μg ml −1 ), MSU (500 μg ml −1 ), CPPD (500 μg ml −1 ) and cystine (500 μg ml −1 ). Cell viability was assessed by MTT assay (a) and cell death was assessed quantifying PI positivity (b) and C shows representative images 24 h later. Original image magnification: × 200, scale bar, 100 μm. ( d ) Mouse tubular epithelial cells were pretreated with RIPK3 inhibitor dabrafenib before exposing to different type crystals. Cell viability was assessed by MTT assay 24 h later. Data are expressed as mean±s.e.m. of three independent experiments, and was analysed using Student's t -test. Baseline viability is set as 100%. * P

    Article Snippet: All cells were stimulated with different doses of crystals of CaOx (1–2 μm size; Alfa aesar, Germany), MSU (25–125 nm size; Invivogen, Germany), CPPD (25–125 nm size; Invivogen, Germany) and cystine (1–2 μm size; Sigma Aldrich, Germany) in different experiments.

    Techniques: Transfection, Sequencing, MTT Assay

    Necroptosis is involved in human acute oxalate nephropathy. ( a – c ) Primary renal human progenitor cells were pretreated with either ZVAD–FMK (10 μM) and Nec-1 (100 μM) or NSA (1 μM) before being exposed to CaOx (1000 μg ml −1 ), MSU (500 μg ml −1 ), CPPD (500 μg ml −1 ) and cystine (500 μg ml −1 ). Cell viability was assessed by MTT assay ( a and b ) and cell death was assessed quantifying PI positivity ( c ) 24 h later. Data are expressed as mean±s.e.m. of three independent experiments. Baseline viability is set as 100%. Data were analysed using Student's t -test. * P

    Journal: Nature Communications

    Article Title: Cytotoxicity of crystals involves RIPK3-MLKL-mediated necroptosis

    doi: 10.1038/ncomms10274

    Figure Lengend Snippet: Necroptosis is involved in human acute oxalate nephropathy. ( a – c ) Primary renal human progenitor cells were pretreated with either ZVAD–FMK (10 μM) and Nec-1 (100 μM) or NSA (1 μM) before being exposed to CaOx (1000 μg ml −1 ), MSU (500 μg ml −1 ), CPPD (500 μg ml −1 ) and cystine (500 μg ml −1 ). Cell viability was assessed by MTT assay ( a and b ) and cell death was assessed quantifying PI positivity ( c ) 24 h later. Data are expressed as mean±s.e.m. of three independent experiments. Baseline viability is set as 100%. Data were analysed using Student's t -test. * P

    Article Snippet: All cells were stimulated with different doses of crystals of CaOx (1–2 μm size; Alfa aesar, Germany), MSU (25–125 nm size; Invivogen, Germany), CPPD (25–125 nm size; Invivogen, Germany) and cystine (1–2 μm size; Sigma Aldrich, Germany) in different experiments.

    Techniques: MTT Assay

    Crystal cytotoxicity involves the necroptosis pathway. ( a ): Protein expression of TNFR1, RIPK1 and RIPK3 was determined by western blot from total proteins isolated 18 h after stimulation of mouse tubular epithelial cells with crystals of CaOx (1,000 μg ml −1 ), MSU (500 μg ml −1 ), CPPD (500 μg ml −1 ) and cystine (500 μg ml −1 ). β-actin was used as loading control. ( b ) Mouse tubular epithelial cells were exposed to different concentrations of CaOx, MSU, CPPD or cystine crystals as indicated in the presence or absence of necrostatin (Nec)-1 (100 μM) together with the pan-caspase inhibitor ZVAD–FMK–FMK (10 μM). Cell viability was assessed by MTT assay 24 h later. Data are expressed as mean cell viability±s.e.m. of three independent experiments. Baseline viability is set as 100%. ( c ) In similar experiments necrotic tubular epithelial cells were identified via propidium iodide positivity and the results were expressed as mean fluorescent intensity on digital analysis of pictures taken from culture dishes. Representative images are shown at an original magnification of × 200, scale bar, 40 μm. Data were analysed using Student's t -test. * P

    Journal: Nature Communications

    Article Title: Cytotoxicity of crystals involves RIPK3-MLKL-mediated necroptosis

    doi: 10.1038/ncomms10274

    Figure Lengend Snippet: Crystal cytotoxicity involves the necroptosis pathway. ( a ): Protein expression of TNFR1, RIPK1 and RIPK3 was determined by western blot from total proteins isolated 18 h after stimulation of mouse tubular epithelial cells with crystals of CaOx (1,000 μg ml −1 ), MSU (500 μg ml −1 ), CPPD (500 μg ml −1 ) and cystine (500 μg ml −1 ). β-actin was used as loading control. ( b ) Mouse tubular epithelial cells were exposed to different concentrations of CaOx, MSU, CPPD or cystine crystals as indicated in the presence or absence of necrostatin (Nec)-1 (100 μM) together with the pan-caspase inhibitor ZVAD–FMK–FMK (10 μM). Cell viability was assessed by MTT assay 24 h later. Data are expressed as mean cell viability±s.e.m. of three independent experiments. Baseline viability is set as 100%. ( c ) In similar experiments necrotic tubular epithelial cells were identified via propidium iodide positivity and the results were expressed as mean fluorescent intensity on digital analysis of pictures taken from culture dishes. Representative images are shown at an original magnification of × 200, scale bar, 40 μm. Data were analysed using Student's t -test. * P

    Article Snippet: All cells were stimulated with different doses of crystals of CaOx (1–2 μm size; Alfa aesar, Germany), MSU (25–125 nm size; Invivogen, Germany), CPPD (25–125 nm size; Invivogen, Germany) and cystine (1–2 μm size; Sigma Aldrich, Germany) in different experiments.

    Techniques: Expressing, Western Blot, Isolation, MTT Assay

    Necrostatin-1 and neutrophil recruitment. ( a , b ): i.p. injection of CaOx, MSU, CPPD or cystine crystals into C57BL/6 mice ( n =4 in each group) induces neutrophil recruitment to the peritoneal cavity as determined by flow cytometry of peritoneal lavage fluids. This process is not affected by concomitant necrostatin-1 treatment. A shows representative flow cytometry data for each crystal presented as mean fluorescence intensity for the neutrophil marker. ( b ) Shows data of each mouse with the mean of wild-type mice set as 100%. NS, not significant. ( c ) Similar experiments using the air pouch model of neutrophil recruitment gave identical results ( n =5 in each group) and are expressed in the same manner. ( d – f ) In vivo microscopy studies of the postischemic musculus cremaster were performed as a second model of injury-associated and ROS-dependent neutrophil recruitment in C57BL/6 mice. Necrostatin-1 significantly reduced the microvascular leakage of FITC-labelled dextran particles as illustrated by representative images in D at an original magnification of × 400, scale bar, 100 μm and quantitatively in ( e ). In the same experiment leukocyte transmigration from the microvasculature was quantified by counting from video recordings taken at baseline and at 60 and 120 min. Data are means±s.e.m. from seven mice in each group. Data were analysed using one-way ANOVA with post hoc Bonferroni's correction. * P

    Journal: Nature Communications

    Article Title: Cytotoxicity of crystals involves RIPK3-MLKL-mediated necroptosis

    doi: 10.1038/ncomms10274

    Figure Lengend Snippet: Necrostatin-1 and neutrophil recruitment. ( a , b ): i.p. injection of CaOx, MSU, CPPD or cystine crystals into C57BL/6 mice ( n =4 in each group) induces neutrophil recruitment to the peritoneal cavity as determined by flow cytometry of peritoneal lavage fluids. This process is not affected by concomitant necrostatin-1 treatment. A shows representative flow cytometry data for each crystal presented as mean fluorescence intensity for the neutrophil marker. ( b ) Shows data of each mouse with the mean of wild-type mice set as 100%. NS, not significant. ( c ) Similar experiments using the air pouch model of neutrophil recruitment gave identical results ( n =5 in each group) and are expressed in the same manner. ( d – f ) In vivo microscopy studies of the postischemic musculus cremaster were performed as a second model of injury-associated and ROS-dependent neutrophil recruitment in C57BL/6 mice. Necrostatin-1 significantly reduced the microvascular leakage of FITC-labelled dextran particles as illustrated by representative images in D at an original magnification of × 400, scale bar, 100 μm and quantitatively in ( e ). In the same experiment leukocyte transmigration from the microvasculature was quantified by counting from video recordings taken at baseline and at 60 and 120 min. Data are means±s.e.m. from seven mice in each group. Data were analysed using one-way ANOVA with post hoc Bonferroni's correction. * P

    Article Snippet: All cells were stimulated with different doses of crystals of CaOx (1–2 μm size; Alfa aesar, Germany), MSU (25–125 nm size; Invivogen, Germany), CPPD (25–125 nm size; Invivogen, Germany) and cystine (1–2 μm size; Sigma Aldrich, Germany) in different experiments.

    Techniques: Injection, Mouse Assay, Flow Cytometry, Cytometry, Fluorescence, Marker, In Vivo, Microscopy, Transmigration Assay

    Crystals induce primary cell necrosis. ( a ): Various crystals were incubated with tubular epithelial cells as indicated. Images show the crystal shapes at a magnification of × 1,000 (left), TEM images shows that these crystals induce necrosis of tubular epithelial cells as indicated by ruptured plasma membranes (middle, × 2,000), scale bar, 2 μm. The images on the right show the same rhodamine-labelled monolayers (red) 24 h later. When Sytox green is added to the medium cells with permeable plasma membranes turn green indicating cell death (× 200), scale bar, 40 μm. ( b ) Flow cytometry was used to define the type and stage of tubular cell (MTC) death on CaOx crystal exposure or ultraviolet light type B for 90 s (s) over a period of 50 h as described in methods. Data for viable cells, apoptotic cells and necrotic cells are expressed as the percentage of viable cells±s.e.m. of all cells for each time point. ( c ): The graphs show a quantitative analysis of the same experiment displaying all different phenotypes of tubular epithelial cells. Flow cytometry cell death definitions are described in the methods section. ( d ) Mouse tubular epithelial cell viability on crystal exposure by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay with and without the pan-caspase inhibitor ZVAD–FMK–FMK. All data are mean±s.e.m. of at least three independent experiments. CaOx, MSU, CPPD, NS, not significant. * P

    Journal: Nature Communications

    Article Title: Cytotoxicity of crystals involves RIPK3-MLKL-mediated necroptosis

    doi: 10.1038/ncomms10274

    Figure Lengend Snippet: Crystals induce primary cell necrosis. ( a ): Various crystals were incubated with tubular epithelial cells as indicated. Images show the crystal shapes at a magnification of × 1,000 (left), TEM images shows that these crystals induce necrosis of tubular epithelial cells as indicated by ruptured plasma membranes (middle, × 2,000), scale bar, 2 μm. The images on the right show the same rhodamine-labelled monolayers (red) 24 h later. When Sytox green is added to the medium cells with permeable plasma membranes turn green indicating cell death (× 200), scale bar, 40 μm. ( b ) Flow cytometry was used to define the type and stage of tubular cell (MTC) death on CaOx crystal exposure or ultraviolet light type B for 90 s (s) over a period of 50 h as described in methods. Data for viable cells, apoptotic cells and necrotic cells are expressed as the percentage of viable cells±s.e.m. of all cells for each time point. ( c ): The graphs show a quantitative analysis of the same experiment displaying all different phenotypes of tubular epithelial cells. Flow cytometry cell death definitions are described in the methods section. ( d ) Mouse tubular epithelial cell viability on crystal exposure by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay with and without the pan-caspase inhibitor ZVAD–FMK–FMK. All data are mean±s.e.m. of at least three independent experiments. CaOx, MSU, CPPD, NS, not significant. * P

    Article Snippet: All cells were stimulated with different doses of crystals of CaOx (1–2 μm size; Alfa aesar, Germany), MSU (25–125 nm size; Invivogen, Germany), CPPD (25–125 nm size; Invivogen, Germany) and cystine (1–2 μm size; Sigma Aldrich, Germany) in different experiments.

    Techniques: Incubation, Transmission Electron Microscopy, Flow Cytometry, Cytometry

    MicroRNA (miR, miRNA)-488 and miR-920 suppress monosodium urate (MSU)-induced expression of proinflammatory cytokines in THP-1 cells. The miRNA mimics or negative control (NC) mimics (50 nM) were transfected into THP-1 cells using Lipofectamine RNAiMAX reagent in accordance with the manufacturer’s instructions. After 24 h of transfection, cells were stimulated for 3 h with 0.5 μM 12-myristate 13-acetate. Then, cells were washed and stimulated with 250 μg/ml MSU crystals for 24 h to detect the production of proinflammatory cytokines. After the treatment, the cells were collected and analyzed by quantitative real-time polymerase chain reaction ( a , c ). The cell culture supernatants were also collected to detect the concentrations of interleukin (IL)-8 and tumor necrosis factor (TNF)-α by enzyme-linked immunosorbent assay ( b , d ). Values are expressed as mean ± SEM of three independent experiments, each of which was run in triplicate. # P

    Journal: Arthritis Research & Therapy

    Article Title: MicroRNA-488 and -920 regulate the production of proinflammatory cytokines in acute gouty arthritis

    doi: 10.1186/s13075-017-1418-6

    Figure Lengend Snippet: MicroRNA (miR, miRNA)-488 and miR-920 suppress monosodium urate (MSU)-induced expression of proinflammatory cytokines in THP-1 cells. The miRNA mimics or negative control (NC) mimics (50 nM) were transfected into THP-1 cells using Lipofectamine RNAiMAX reagent in accordance with the manufacturer’s instructions. After 24 h of transfection, cells were stimulated for 3 h with 0.5 μM 12-myristate 13-acetate. Then, cells were washed and stimulated with 250 μg/ml MSU crystals for 24 h to detect the production of proinflammatory cytokines. After the treatment, the cells were collected and analyzed by quantitative real-time polymerase chain reaction ( a , c ). The cell culture supernatants were also collected to detect the concentrations of interleukin (IL)-8 and tumor necrosis factor (TNF)-α by enzyme-linked immunosorbent assay ( b , d ). Values are expressed as mean ± SEM of three independent experiments, each of which was run in triplicate. # P

    Article Snippet: MSU crystals can induce a variety of cytokines, including IL-1β, IL-6, IL-8, and TNF-α [ ].

    Techniques: Expressing, Negative Control, Transfection, Real-time Polymerase Chain Reaction, Cell Culture, Enzyme-linked Immunosorbent Assay

    Monosodium urate (MSU) crystals promote the expression of proinflammatory cytokines in THP-1 cells. THP-1 cells were stimulated by the indicated concentration of MSU crystals. The messenger (mRNA) expression of interleukin (IL)-1β, IL-8, and tumor necrosis factor (TNF)-α were detected by quantitative real-time polymerase chain reaction ( a – c ). Protein expression of IL-1β, IL-8, and TNF-α was detected by enzyme-linked immunosorbent assay ( d – f ). Values are expressed as mean ± SEM of three independent experiments, each of which was run in triplicate. * P

    Journal: Arthritis Research & Therapy

    Article Title: MicroRNA-488 and -920 regulate the production of proinflammatory cytokines in acute gouty arthritis

    doi: 10.1186/s13075-017-1418-6

    Figure Lengend Snippet: Monosodium urate (MSU) crystals promote the expression of proinflammatory cytokines in THP-1 cells. THP-1 cells were stimulated by the indicated concentration of MSU crystals. The messenger (mRNA) expression of interleukin (IL)-1β, IL-8, and tumor necrosis factor (TNF)-α were detected by quantitative real-time polymerase chain reaction ( a – c ). Protein expression of IL-1β, IL-8, and TNF-α was detected by enzyme-linked immunosorbent assay ( d – f ). Values are expressed as mean ± SEM of three independent experiments, each of which was run in triplicate. * P

    Article Snippet: MSU crystals can induce a variety of cytokines, including IL-1β, IL-6, IL-8, and TNF-α [ ].

    Techniques: Expressing, Concentration Assay, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    Time and treatment-dependent phagocytosis of monosodium urate monohydrate (MSU) crystals by differentiated human THP-1 macrophages using flow cytometry and impact of recombinant human proteoglycan-4 (rhPRG4) or bovine submaxillary mucin (BSM) treatments following 2 and 4-h incubations. Quantitative determination of MSU phagocytosis was performed using the percentage of cells in the P2 region of interest. Data represent the mean ± S.D. of four independent experiments. * p

    Journal: Arthritis Research & Therapy

    Article Title: Recombinant human proteoglycan-4 reduces phagocytosis of urate crystals and downstream nuclear factor kappa B and inflammasome activation and production of cytokines and chemokines in human and murine macrophages

    doi: 10.1186/s13075-018-1693-x

    Figure Lengend Snippet: Time and treatment-dependent phagocytosis of monosodium urate monohydrate (MSU) crystals by differentiated human THP-1 macrophages using flow cytometry and impact of recombinant human proteoglycan-4 (rhPRG4) or bovine submaxillary mucin (BSM) treatments following 2 and 4-h incubations. Quantitative determination of MSU phagocytosis was performed using the percentage of cells in the P2 region of interest. Data represent the mean ± S.D. of four independent experiments. * p

    Article Snippet: THP-1 macrophages were treated with endotoxin-free MSU crystals (100μg/ml; Invivogen, USA) ± bovine submaxillary mucin (BSM; molecular mass > 1000 KDa) (Sigma Aldrich) (25 μg/ml) or rhPRG4 (molecular mass is approximately 240 KDa) (100 μg/ml) for 2 and 4 h at 37 °C. rhPRG4 is an endotoxin-free full-length product produced by CHO-M cells (Lubris, Framingham, MA, USA) [ ].

    Techniques: Flow Cytometry, Cytometry, Recombinant

    Impact of recombinant human proteoglycan-4 (rhPRG4) treatment on monosodium urate monohydrate (MSU) crystal-induced expression and production of proinflammatory cytokines and chemokines and nuclear factor kappa b (NFκB) p65 subunit nuclear translocation in THP-1 macrophages. Cytokines included interleukin-1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α). Chemokines included interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1). Gene expression data are presented as fold induction of proinflammatory cytokines and chemokines gene expression compared to control untreated THP-1 macrophages. THP-1 macrophages were treated with MSU crystals (100μg/ml) ± rhPRG4 (100μg/ml) or bovine submaxillary mucin (BSM; 25μg/ml) for 6 h ( a through d ). NFκB p65 subunit nuclear translocation in THP-1 macrophages was performed at 1 h following MSU challenge (100μg/ml). Gene expression studies were performed at 6 h ( f through i ) and cytokine and chemokine media concentrations were determined at 24 h ( j through m ). Data represent the mean ± S.D. of three to four independent experiments with duplicate wells per group. *p

    Journal: Arthritis Research & Therapy

    Article Title: Recombinant human proteoglycan-4 reduces phagocytosis of urate crystals and downstream nuclear factor kappa B and inflammasome activation and production of cytokines and chemokines in human and murine macrophages

    doi: 10.1186/s13075-018-1693-x

    Figure Lengend Snippet: Impact of recombinant human proteoglycan-4 (rhPRG4) treatment on monosodium urate monohydrate (MSU) crystal-induced expression and production of proinflammatory cytokines and chemokines and nuclear factor kappa b (NFκB) p65 subunit nuclear translocation in THP-1 macrophages. Cytokines included interleukin-1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α). Chemokines included interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1). Gene expression data are presented as fold induction of proinflammatory cytokines and chemokines gene expression compared to control untreated THP-1 macrophages. THP-1 macrophages were treated with MSU crystals (100μg/ml) ± rhPRG4 (100μg/ml) or bovine submaxillary mucin (BSM; 25μg/ml) for 6 h ( a through d ). NFκB p65 subunit nuclear translocation in THP-1 macrophages was performed at 1 h following MSU challenge (100μg/ml). Gene expression studies were performed at 6 h ( f through i ) and cytokine and chemokine media concentrations were determined at 24 h ( j through m ). Data represent the mean ± S.D. of three to four independent experiments with duplicate wells per group. *p

    Article Snippet: THP-1 macrophages were treated with endotoxin-free MSU crystals (100μg/ml; Invivogen, USA) ± bovine submaxillary mucin (BSM; molecular mass > 1000 KDa) (Sigma Aldrich) (25 μg/ml) or rhPRG4 (molecular mass is approximately 240 KDa) (100 μg/ml) for 2 and 4 h at 37 °C. rhPRG4 is an endotoxin-free full-length product produced by CHO-M cells (Lubris, Framingham, MA, USA) [ ].

    Techniques: Recombinant, Expressing, Translocation Assay

    Impact of recombinant human proteoglycan-4 (rhPRG4) treatment on monosodium urate monohydrate (MSU) crystal-induced NLRP3 inflammasome activation in THP-1 macrophages. THP-1 macrophages were treated with 100μg/ml MSU in the absence or presence of rhPRG4 (100 and 200μg/ml) for 12 h. H 2 O 2 (5 mM) was used as a positive control. Data represent the mean ± S.D. of three independent experiments. * p

    Journal: Arthritis Research & Therapy

    Article Title: Recombinant human proteoglycan-4 reduces phagocytosis of urate crystals and downstream nuclear factor kappa B and inflammasome activation and production of cytokines and chemokines in human and murine macrophages

    doi: 10.1186/s13075-018-1693-x

    Figure Lengend Snippet: Impact of recombinant human proteoglycan-4 (rhPRG4) treatment on monosodium urate monohydrate (MSU) crystal-induced NLRP3 inflammasome activation in THP-1 macrophages. THP-1 macrophages were treated with 100μg/ml MSU in the absence or presence of rhPRG4 (100 and 200μg/ml) for 12 h. H 2 O 2 (5 mM) was used as a positive control. Data represent the mean ± S.D. of three independent experiments. * p

    Article Snippet: THP-1 macrophages were treated with endotoxin-free MSU crystals (100μg/ml; Invivogen, USA) ± bovine submaxillary mucin (BSM; molecular mass > 1000 KDa) (Sigma Aldrich) (25 μg/ml) or rhPRG4 (molecular mass is approximately 240 KDa) (100 μg/ml) for 2 and 4 h at 37 °C. rhPRG4 is an endotoxin-free full-length product produced by CHO-M cells (Lubris, Framingham, MA, USA) [ ].

    Techniques: Recombinant, Activation Assay, Positive Control

    iNKT Cells Induce IL-1β Secretion by BMDCs in a CD1d-Dependent Manner (A and B) BMDCs were left unprimed or primed with Pam3CSK4 (P3C; 0.5 μg/mL) for 24 h and subsequently co-cultured with iNKT cells for 24 h in the (A) absence or (B) presence of αGalCer (50 ng/mL). IL-1β release was quantified by ELISA. As a control, BMDCs were primed with P3C for 4 h, followed by 6-h stimulation with MSU (100 μg/mL). The same concentrations of P3C, αGalCer, and MSU were used throughout all experiments. (C and D) P3C-primed BMDCs were cultured alone or with iNKT cells for the indicated times in the presence or absence of αGalCer. IL-1β release was quantified by ELISA (C), and cell-associated and extracellular IL-1β were analyzed by immunoblot (D). As a control, BMDCs were primed with P3C for 4 h, followed by 1-h stimulation with nigericin (10 μM). (E) P3C-primed BMDCs from WT and CD1d −/− mice were co-cultured with iNKT cells for the indicated times in the absence or presence of αGalCer. IL-1β release was quantified by ELISA. P3C-primed BMDCs stimulated with nigericin were used as a control. Error bars represent mean + SD of triplicate samples. Each panel is representative of at least three different experiments. *p

    Journal: Cell reports

    Article Title: A Two-Cell Model for IL-1β Release Mediated by Death-Receptor Signaling

    doi: 10.1016/j.celrep.2020.03.030

    Figure Lengend Snippet: iNKT Cells Induce IL-1β Secretion by BMDCs in a CD1d-Dependent Manner (A and B) BMDCs were left unprimed or primed with Pam3CSK4 (P3C; 0.5 μg/mL) for 24 h and subsequently co-cultured with iNKT cells for 24 h in the (A) absence or (B) presence of αGalCer (50 ng/mL). IL-1β release was quantified by ELISA. As a control, BMDCs were primed with P3C for 4 h, followed by 6-h stimulation with MSU (100 μg/mL). The same concentrations of P3C, αGalCer, and MSU were used throughout all experiments. (C and D) P3C-primed BMDCs were cultured alone or with iNKT cells for the indicated times in the presence or absence of αGalCer. IL-1β release was quantified by ELISA (C), and cell-associated and extracellular IL-1β were analyzed by immunoblot (D). As a control, BMDCs were primed with P3C for 4 h, followed by 1-h stimulation with nigericin (10 μM). (E) P3C-primed BMDCs from WT and CD1d −/− mice were co-cultured with iNKT cells for the indicated times in the absence or presence of αGalCer. IL-1β release was quantified by ELISA. P3C-primed BMDCs stimulated with nigericin were used as a control. Error bars represent mean + SD of triplicate samples. Each panel is representative of at least three different experiments. *p

    Article Snippet: For inflammasome activation controls, BMDCs were primed for 4 hours and treated with monosodium urate (MSU) crystals (Invivogen, 100 μg/ml) for 6 hours or nigericin (Invivogen, 10 μM) for 1 hour.

    Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Mouse Assay

    iNKT Cells Employ the Death Receptor Ligand FasL to Instruct TLR-Primed BMDCs to Release IL-1β (A) iNKT cells were cultured in the presence of increasing concentrations of plate-bound anti-CD3 for 24 h, after which their cell-free Supes were used to stimulate P3C-primed BMDCs for 24 h. IL-1β (left) and interferon γ (IFN-γ; right) in the Supes were quantified by ELISA. Nigericin-stimulated BMDCs were used as a control. (B) The ImmGen database was mined to compare the expression of cell-surface proteins (Gene Ontology term “cell surface,” GO0009986) expressed by iNKT cells versus other αβ T cells. The red dots represent genes expressed by iNKT cells with a fold change of greater than 5 compared with all other αβ T cells. Expression values comparing class means for specific T cell populations are shown in the right panel. (C) P3C-primed BMDCs were co-cultured with iNKT cells for 24 h in the presence of a FasL-blocking antibody (aFasL mAb) or an isotype control. IL-1β release was quantified by ELISA. MSU-treated BMDCs were used as a control. (D) P3C-primed BMDCs from WT and Fas −/− mice were co-cultured with iNKT cells for 24 h. IL-1β release was quantified by ELISA. MSU-treated BMDCs were used as a control. (E) Resting iNKT cells were stained with a FasL-specific antibody or an isotype control and subsequently analyzed by flow cytometry. The left panel represents surface staining, whereas the right panel represents permeabilized iNKT cells. (F) P3C-primed BMDCs from WT and CD1d −/− mice were co-cultured with CellTrace Violet-labeled-iNKT cells for the indicated times in the presence or absence of αGalCer, and the expression of FasL on the surface of iNKT cells was analyzed by flow cytometry. Numbers on the right of each histogram represent mean fluorescence intensity values. Error bars represent mean + SD of triplicate samples. Each panel is representative of at least three different experiments.

    Journal: Cell reports

    Article Title: A Two-Cell Model for IL-1β Release Mediated by Death-Receptor Signaling

    doi: 10.1016/j.celrep.2020.03.030

    Figure Lengend Snippet: iNKT Cells Employ the Death Receptor Ligand FasL to Instruct TLR-Primed BMDCs to Release IL-1β (A) iNKT cells were cultured in the presence of increasing concentrations of plate-bound anti-CD3 for 24 h, after which their cell-free Supes were used to stimulate P3C-primed BMDCs for 24 h. IL-1β (left) and interferon γ (IFN-γ; right) in the Supes were quantified by ELISA. Nigericin-stimulated BMDCs were used as a control. (B) The ImmGen database was mined to compare the expression of cell-surface proteins (Gene Ontology term “cell surface,” GO0009986) expressed by iNKT cells versus other αβ T cells. The red dots represent genes expressed by iNKT cells with a fold change of greater than 5 compared with all other αβ T cells. Expression values comparing class means for specific T cell populations are shown in the right panel. (C) P3C-primed BMDCs were co-cultured with iNKT cells for 24 h in the presence of a FasL-blocking antibody (aFasL mAb) or an isotype control. IL-1β release was quantified by ELISA. MSU-treated BMDCs were used as a control. (D) P3C-primed BMDCs from WT and Fas −/− mice were co-cultured with iNKT cells for 24 h. IL-1β release was quantified by ELISA. MSU-treated BMDCs were used as a control. (E) Resting iNKT cells were stained with a FasL-specific antibody or an isotype control and subsequently analyzed by flow cytometry. The left panel represents surface staining, whereas the right panel represents permeabilized iNKT cells. (F) P3C-primed BMDCs from WT and CD1d −/− mice were co-cultured with CellTrace Violet-labeled-iNKT cells for the indicated times in the presence or absence of αGalCer, and the expression of FasL on the surface of iNKT cells was analyzed by flow cytometry. Numbers on the right of each histogram represent mean fluorescence intensity values. Error bars represent mean + SD of triplicate samples. Each panel is representative of at least three different experiments.

    Article Snippet: For inflammasome activation controls, BMDCs were primed for 4 hours and treated with monosodium urate (MSU) crystals (Invivogen, 100 μg/ml) for 6 hours or nigericin (Invivogen, 10 μM) for 1 hour.

    Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Expressing, Blocking Assay, Mouse Assay, Staining, Flow Cytometry, Labeling, Fluorescence

    Gasdermin D pore formation and pyroptosis are not involved in EEA1 release. ( A ) Immunoblot analysis of EEA1 and IL-1β on cell lysate and cell-free supernatant from LPS-primed BMDMs from wild type or Casp1/11 −/− after stimulation with nigericin, ATP, E . coli or MSU crystals as indicated. IL-1β released to the extracellular medium was detected by ELISA (bottom). ( B ) LDH release from macrophages treated as in A. ( C ) Immunoblot analysis of EEA1 on cell lysate and cell-free supernatant from BMDMs wild-type and Gsdmd −/− after nigericin stimulation. ( D ) Immunoblot analysis of caspase-1 and IL-1β on cell lysate and cell-free supernatant from wild-type or Gsdmd −/− BMDMs after nigericin stimulation in the presence or absence of the caspase-1 inhibitor Ac-YVAD. ( E , F ) ELISA for released IL-1β ( E ) or LDH ( F ) from macrophages treated as in D. ( G ) Immunoblot analysis of ASC and NLRP3 on cell-free supernatant from macrophages stimulated as in D. ( H ) Yo-Pro uptake in macrophages treated as in D. Blots are representative of three to four independent experiments. Nitrocellulose membranes were cropped after protein blotting and then inmunodetection was carried out separately for each antibody. Complete blots for IL-1β and Caspase-1 are showed as supplementary. Data are presented as average ± sem from n = 3 independent experiments.

    Journal: Scientific Reports

    Article Title: Early endosome autoantigen 1 regulates IL-1β release upon caspase-1 activation independently of gasdermin D membrane permeabilization

    doi: 10.1038/s41598-019-42298-4

    Figure Lengend Snippet: Gasdermin D pore formation and pyroptosis are not involved in EEA1 release. ( A ) Immunoblot analysis of EEA1 and IL-1β on cell lysate and cell-free supernatant from LPS-primed BMDMs from wild type or Casp1/11 −/− after stimulation with nigericin, ATP, E . coli or MSU crystals as indicated. IL-1β released to the extracellular medium was detected by ELISA (bottom). ( B ) LDH release from macrophages treated as in A. ( C ) Immunoblot analysis of EEA1 on cell lysate and cell-free supernatant from BMDMs wild-type and Gsdmd −/− after nigericin stimulation. ( D ) Immunoblot analysis of caspase-1 and IL-1β on cell lysate and cell-free supernatant from wild-type or Gsdmd −/− BMDMs after nigericin stimulation in the presence or absence of the caspase-1 inhibitor Ac-YVAD. ( E , F ) ELISA for released IL-1β ( E ) or LDH ( F ) from macrophages treated as in D. ( G ) Immunoblot analysis of ASC and NLRP3 on cell-free supernatant from macrophages stimulated as in D. ( H ) Yo-Pro uptake in macrophages treated as in D. Blots are representative of three to four independent experiments. Nitrocellulose membranes were cropped after protein blotting and then inmunodetection was carried out separately for each antibody. Complete blots for IL-1β and Caspase-1 are showed as supplementary. Data are presented as average ± sem from n = 3 independent experiments.

    Article Snippet: Reagents E . coli LPS O55:B5, DAPI, ATP, triton X-100, nigericin, anti-V5 antibody, PMA and puromycin resistant MISSION® shRNA Lentiviral Transduction Particles were from Sigma-Aldrich; Recombinant human caspase-1 and caspase-1 inhibitor Ac-YVAD-AOM were from Merk-Millipore; MSU crystals were from Enzo Life Sciences; flagellin from S . typhimurium from Invivogen; the receptor-binding protein protective antigen and metalloprotease lethal factor were from List Biological; the protein-delivery reagent PULSin was from PolyPlus Transfection;Yo-Pro-1 from Life Technologies.

    Techniques: Enzyme-linked Immunosorbent Assay

    Four axial spondyloarthritis (AxSpA) patients with monosodium urate (MSU) crystal and radiographic structural damage at the sacroiliac joint. For each set of images, panel a shows the sacroiliac joint on plain radiographs, panel b shows the three-dimensional reconstruction dual-energy computed tomography (DECT) images, panel c shows the corresponding coronal (patient 2 and 4) or axial (patient 1 and 3) DECT images, and panel d shows the corresponding level of computed tomography (CT) images. A large quantity of MSU crystal deposition shown as green was found at the sacroiliac joint or the surrounding area in the DECT images. Four male patients (patient 1–4), aged 36, 44, 23, and 27 years old, had serum uric acid levels of 407 μmol/L, 370 μmol/L, 572 μmol/L, and 464 μmol/L, respectively. They were graded with a scale of 0, 0, II, III at the left sacroiliac joint and 0, I, II, III at the right sacroiliac joint, respectively, on plain radiographs

    Journal: Arthritis Research & Therapy

    Article Title: Monosodium urate crystal deposition associated with the progress of radiographic grade at the sacroiliac joint in axial SpA: a dual-energy CT study

    doi: 10.1186/s13075-017-1286-0

    Figure Lengend Snippet: Four axial spondyloarthritis (AxSpA) patients with monosodium urate (MSU) crystal and radiographic structural damage at the sacroiliac joint. For each set of images, panel a shows the sacroiliac joint on plain radiographs, panel b shows the three-dimensional reconstruction dual-energy computed tomography (DECT) images, panel c shows the corresponding coronal (patient 2 and 4) or axial (patient 1 and 3) DECT images, and panel d shows the corresponding level of computed tomography (CT) images. A large quantity of MSU crystal deposition shown as green was found at the sacroiliac joint or the surrounding area in the DECT images. Four male patients (patient 1–4), aged 36, 44, 23, and 27 years old, had serum uric acid levels of 407 μmol/L, 370 μmol/L, 572 μmol/L, and 464 μmol/L, respectively. They were graded with a scale of 0, 0, II, III at the left sacroiliac joint and 0, I, II, III at the right sacroiliac joint, respectively, on plain radiographs

    Article Snippet: Reproducibility of DECT MSU crystal volume measurement and correlation between the average MSU crystal volume and serum uric acid Of the patients who showed the presence of MSU crystal deposition, 57 showed deposition at the left sacroiliac joint, 54 at the right sacroiliac joint, and 154 at the pelvis.

    Techniques: Computed Tomography

    Bland-Altman plots for interobserver reproducibility analysis. a The dual-energy computed tomography (DECT) monosodium urate (MSU) crystal volume at the left sacroiliac joint. b The DECT MSU crystal volume at the right sacroiliac joint. c The DECT MSU crystal volume at the pelvis. Solid line shows bias and dashed lines show the 95% limits of agreement

    Journal: Arthritis Research & Therapy

    Article Title: Monosodium urate crystal deposition associated with the progress of radiographic grade at the sacroiliac joint in axial SpA: a dual-energy CT study

    doi: 10.1186/s13075-017-1286-0

    Figure Lengend Snippet: Bland-Altman plots for interobserver reproducibility analysis. a The dual-energy computed tomography (DECT) monosodium urate (MSU) crystal volume at the left sacroiliac joint. b The DECT MSU crystal volume at the right sacroiliac joint. c The DECT MSU crystal volume at the pelvis. Solid line shows bias and dashed lines show the 95% limits of agreement

    Article Snippet: Reproducibility of DECT MSU crystal volume measurement and correlation between the average MSU crystal volume and serum uric acid Of the patients who showed the presence of MSU crystal deposition, 57 showed deposition at the left sacroiliac joint, 54 at the right sacroiliac joint, and 154 at the pelvis.

    Techniques: Computed Tomography

    Cytokine induction in early infection does not involve an autocrine mechanism. (A) After incubation with the indicated amount of recombinant human IL-1Ra for 30 min, miCtrl cells were stimulated with recombinant human IL-1β for 60 min, and the IL-8 secretion was analyzed by ELISA. (B) After incubation with 100 ng/ml IL-1Ra for 30 min, miCtrl cells were infected with S . aureus at an MOI of 4 for the times indicated. IL-8 and TNF-α secretion from each cell line were analyzed by ELISA. (C) After incubation with 20 μM Ac-YVAD-CMK for 30 min, Dox-untreated miCtrl and miNLRP3 cells were infected with S . aureus for 100 min. TNF-α and IL-1β secretion were analyzed by ELISA. (D) THP-1 cells were incubated with 5 μg/ml CHX for 30 min and then infected with S . aureus at an MOI of 4 for the indicated times. TNF-α and IL-1β secretion were analyzed by ELISA. (E) THP-1 cells were treated with CHX and infected as in (D). The total RNA was analyzed for TNF-α and IL-1β mRNA by RT-PCR. (F) ELISA analysis of TNF-α and IL-1β released from THP-1 cells treated with MSU in the presence or absence of 0.63, 1.25, 2.5, 5, and 10 μM Ac-YVAD-CMK for 180 min. (G) ELISA analysis of IL-18 released from THP-1 cells treated as in (C) (left panel) or treated with MSU in the presence or absence of 10 μM of Ac-YVAD-CMK for 20 h (right panel). All results are representative of three independent experiments. ND, not detected.

    Journal: PLoS ONE

    Article Title: NLRP3 Mediates NF-κB Activation and Cytokine Induction in Microbially Induced and Sterile Inflammation

    doi: 10.1371/journal.pone.0119179

    Figure Lengend Snippet: Cytokine induction in early infection does not involve an autocrine mechanism. (A) After incubation with the indicated amount of recombinant human IL-1Ra for 30 min, miCtrl cells were stimulated with recombinant human IL-1β for 60 min, and the IL-8 secretion was analyzed by ELISA. (B) After incubation with 100 ng/ml IL-1Ra for 30 min, miCtrl cells were infected with S . aureus at an MOI of 4 for the times indicated. IL-8 and TNF-α secretion from each cell line were analyzed by ELISA. (C) After incubation with 20 μM Ac-YVAD-CMK for 30 min, Dox-untreated miCtrl and miNLRP3 cells were infected with S . aureus for 100 min. TNF-α and IL-1β secretion were analyzed by ELISA. (D) THP-1 cells were incubated with 5 μg/ml CHX for 30 min and then infected with S . aureus at an MOI of 4 for the indicated times. TNF-α and IL-1β secretion were analyzed by ELISA. (E) THP-1 cells were treated with CHX and infected as in (D). The total RNA was analyzed for TNF-α and IL-1β mRNA by RT-PCR. (F) ELISA analysis of TNF-α and IL-1β released from THP-1 cells treated with MSU in the presence or absence of 0.63, 1.25, 2.5, 5, and 10 μM Ac-YVAD-CMK for 180 min. (G) ELISA analysis of IL-18 released from THP-1 cells treated as in (C) (left panel) or treated with MSU in the presence or absence of 10 μM of Ac-YVAD-CMK for 20 h (right panel). All results are representative of three independent experiments. ND, not detected.

    Article Snippet: Cycloheximide (CHX) and MSU crystals were purchased from Wako Pure Chemical Industries (Osaka, Japan).

    Techniques: Infection, Incubation, Recombinant, Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction

    IL-1β secretion by PBMCs following exposure to MSU crystals or SNPs. Secretion of IL-1β by PBMCs with (main figure) or without (inset) initial exposure to 10 ng/mL LPS for 3 h followed by exposure to MSU crystals (100 μg/mL) or SNPs (100 μg/mL) for a further 3 h. The supernatant was either collected immediately for analysis (3 h; open bars) or following a further 21 h of cell incubation in fresh (i.e. without added particles or MAMPs) TCM (3 + 21 h; black bars). All data are expressed as mean ± SEM ( n = 4). Results were analysed by paired T test in comparison to baseline (B), i.e. non-particle-exposed cells; * p

    Journal: Particle and Fibre Toxicology

    Article Title: Pro-inflammatory adjuvant properties of pigment-grade titanium dioxide particles are augmented by a genotype that potentiates interleukin 1β processing

    doi: 10.1186/s12989-017-0232-2

    Figure Lengend Snippet: IL-1β secretion by PBMCs following exposure to MSU crystals or SNPs. Secretion of IL-1β by PBMCs with (main figure) or without (inset) initial exposure to 10 ng/mL LPS for 3 h followed by exposure to MSU crystals (100 μg/mL) or SNPs (100 μg/mL) for a further 3 h. The supernatant was either collected immediately for analysis (3 h; open bars) or following a further 21 h of cell incubation in fresh (i.e. without added particles or MAMPs) TCM (3 + 21 h; black bars). All data are expressed as mean ± SEM ( n = 4). Results were analysed by paired T test in comparison to baseline (B), i.e. non-particle-exposed cells; * p

    Article Snippet: Finally, paired T tests were used to compare supernatant levels of IL-1β for cells exposed to MSU crystals or SNPs versus non-particle-exposed control cells.

    Techniques: Incubation

    Extracellular succinate signals via GPR91 to stimulate macrophages to release IL-1β. (A) GPR91 mRNA expression in WT (Janvier C57BL/6J) inflammatory BMDMs (M-CSF + IFN-γ) ± 100 ng/ml LPS, 500 µM succinate, or 10 ng/ml IL-1β for 24 h. n = 3 of Ct values. Succinate (Succ), IL-1β, and LPS related to basal (=1). Data are representative of three experiments. (B) Succinate levels (mass spectrophotometry area ratio) in medium from cultured BMDMs. Extracellular succinate from WT (littermates; black bars) and Sucnr1 −/− (gray bars), neutral (M, M-CSF), or inflammatory (M + IFN-γ) BMDMs ± 100 ng/ml LPS for 24 h is shown. n = 6 wells. Data are representative of three experiments. (C) IL-1β in supernatants of WT (Janvier C57BL/6J) and Sucnr1 −/− neutral or inflammatory BMDMs ± 100 ng/ml LPS for 24 h. n = 3 wells and are representative of seven experiments. (D) IL-1β mRNA levels from cell lysates from WT (Janvier C57BL/6J) or Sucnr1 −/− inflammatory BMDMs ± 100 ng/ml LPS at 4 h (related to WT basal = 1). n = 2–3 of Ct values. Data are representative of two experiments. (E) IL-1β levels measured in the supernatant of WT (Janvier C57BL/6J) and Sucnr1 −/− inflammatory BMDMs stimulated with 1 ng/ml LPS and 180 µg/ml MSU. n = 5–6 wells. Data are representative of two experiments. (F) Western blot of HIF-1α (representative blot of two experiments) and quantification (two experiments; 100% for no stimulus, WT, and Sucnr1 −/− ) at 6 h after stimulation with 500 µM succinate, 100 ng/ml LPS, or a combination of the two in WT littermate controls and Sucnr1 −/− inflammatory BMDMs. *, P

    Journal: The Journal of Experimental Medicine

    Article Title: GPR91 senses extracellular succinate released from inflammatory macrophages and exacerbates rheumatoid arthritis

    doi: 10.1084/jem.20160061

    Figure Lengend Snippet: Extracellular succinate signals via GPR91 to stimulate macrophages to release IL-1β. (A) GPR91 mRNA expression in WT (Janvier C57BL/6J) inflammatory BMDMs (M-CSF + IFN-γ) ± 100 ng/ml LPS, 500 µM succinate, or 10 ng/ml IL-1β for 24 h. n = 3 of Ct values. Succinate (Succ), IL-1β, and LPS related to basal (=1). Data are representative of three experiments. (B) Succinate levels (mass spectrophotometry area ratio) in medium from cultured BMDMs. Extracellular succinate from WT (littermates; black bars) and Sucnr1 −/− (gray bars), neutral (M, M-CSF), or inflammatory (M + IFN-γ) BMDMs ± 100 ng/ml LPS for 24 h is shown. n = 6 wells. Data are representative of three experiments. (C) IL-1β in supernatants of WT (Janvier C57BL/6J) and Sucnr1 −/− neutral or inflammatory BMDMs ± 100 ng/ml LPS for 24 h. n = 3 wells and are representative of seven experiments. (D) IL-1β mRNA levels from cell lysates from WT (Janvier C57BL/6J) or Sucnr1 −/− inflammatory BMDMs ± 100 ng/ml LPS at 4 h (related to WT basal = 1). n = 2–3 of Ct values. Data are representative of two experiments. (E) IL-1β levels measured in the supernatant of WT (Janvier C57BL/6J) and Sucnr1 −/− inflammatory BMDMs stimulated with 1 ng/ml LPS and 180 µg/ml MSU. n = 5–6 wells. Data are representative of two experiments. (F) Western blot of HIF-1α (representative blot of two experiments) and quantification (two experiments; 100% for no stimulus, WT, and Sucnr1 −/− ) at 6 h after stimulation with 500 µM succinate, 100 ng/ml LPS, or a combination of the two in WT littermate controls and Sucnr1 −/− inflammatory BMDMs. *, P

    Article Snippet: In vitro stimulation of mouse macrophages or U937 cells U937 or mouse macrophages derived from 7–9-wk-old age-matched animals were incubated for up to 24 h in 96-well flat-bottom plates at a density of 105 cells per well in RPMI-1640 containing 1 or 100 ng/ml LPS (InvivoGen), 500 µM succinate (sodium succinate dibasic hexahydrate; Sigma-Aldrich), 10 ng/ml mouse IL-1β (PeproTech), 180 µg/ml MSU (Enzo Life Sciences), 5 µM GPR91 antagonist GPR91A1 (synthesized in house from ), and 10% human RA SFs (Asterand).

    Techniques: Expressing, Spectrophotometry, Cell Culture, Western Blot