Journal: The Journal of Experimental Medicine

Article Title: An inherited mutation in NLRC4 causes autoinflammation in human and mice

doi: 10.1084/jem.20141091

Figure Lengend Snippet: A mutation in NLRC4 induces overproduction of IL-1β through activation of caspase-1. (a) Flag-tagged wild-type, mutant NLRC4 expression vectors or empty vector (EV) were transfected into 293T cells. 2 d later, after the cell lysates were subjected to SDS-PAGE, Western blotting was performed with an anti-Flag or anti-actin antibody. Red triangle indicates NLRC4. The data in the figure are representative of three independent experiments. (b) Cell lysates from 293T cells transfected with empty vector (EV), flag-tagged wild-type, or mutant NLRC4 were subjected to Blue Native PAGE and the expression of the tagged proteins or actin was evaluated by Western blotting with an anti-Flag or actin antibody, respectively. The data in the figure are representative of three independent experiments. (c) 293T cells were transfected with a CASP1 expression vector together with empty vector (EV), an expression vector for wild-type or mutant NLRC4 . 24 h later, the expression of procaspase-1, cleaved active caspase-1 (10 kD), and actin was evaluated by Western blotting. Red triangles indicate procaspase-1, cleaved active caspase-1, and actin. The data in the figure are representative of five independent experiments. (d) 293T cells were transfected with expression vectors for CASP1 and IL1B together with empty vector (EV), an expression vector for wild-type or mutant NLRC4 . 24 h later, the concentration of IL-1β in the supernatant was evaluated by ELISA. The data are shown as means ± SD (**, P < 0.01; n = 5). The data in the figure are representative of three independent experiments. (e) Peripheral blood mononuclear cells were transfected with three concentrations of Prgl and cultured for 48 h. The concentrations of IL-1β in the supernatants were measured by ELISA. The data shown are means ± SD (**, P < 0.01). The data in the figure are representative of three independent experiments.

Article Snippet: The concentration of G-CSF was measured with the Mouse G-CSF Quantikine ELISA kit (R&D Systems).

Techniques: Mutagenesis, Activation Assay, Expressing, Plasmid Preparation, Transfection, SDS Page, Western Blot, Blue Native PAGE, Concentration Assay, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: The Journal of Experimental Medicine

Article Title: An inherited mutation in NLRC4 causes autoinflammation in human and mice

doi: 10.1084/jem.20141091

Figure Lengend Snippet: BM-derived macrophages that overexpress mutant Nlrc4 secrete IL-1β. BM-derived macrophages (BMMC) infected with control retrovirus, retroviruses encoding wild-type, or mutant Nlrc4 were stimulated with 10 µg/ml LPS for 1 d in the absence (black) or presence (gray) of a caspase-1 inhibitor. As the control, unstimulated BMMCs were used (white). The concentrations of IL-1β and IL-6 in the supernatant were measured by ELISA. The data shown are means ± SD (*, P < 0.05; n = 5). The data shown in this figure are representative of three independent experiments.

Article Snippet: The concentration of G-CSF was measured with the Mouse G-CSF Quantikine ELISA kit (R&D Systems).

Techniques: Derivative Assay, Mutagenesis, Infection, Control, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Experimental Medicine

Article Title: An inherited mutation in NLRC4 causes autoinflammation in human and mice

doi: 10.1084/jem.20141091

Figure Lengend Snippet: Cytokine production in mu-Nlrc4 mice or mice reconstituted with mutant Nlrc4 transduced BM cells. (a) Splenocytes from wild-type or mu-Nlrc4 mice were stimulated with LPS for 24 h, and the concentration of IL-1β in each supernatant was evaluated by ELISA. The data shown are means ± SD (**, P < 0.01). N.D.: not detected. The data in the figure are representative of three independent experiments. (b) The concentrations of IL-1β, IL-17A, and G-CSF in the serum of wild-type (white) and mu-Nlrc4 (black) mice at the age of 8 wk were evaluated by ELISA. For all panels, the data shown are means ± SD (**, P < 0.01; n = 5). N.D.: not detected. The data in the figure are representative of three independent experiments. (c) Intraperitoneal cells or splenocytes from C57BL/6 mice transplanted with BM cells infected with control virus (white), wild-type (black), or mutant Nlrc4 (gray)-encoding virus were evaluated for the number of Gr1 + CD11b + cells 2 mo after the transplantation. The data shown are means ± SD (*, P < 0.05; n = 5 in each group). The data in the figure are representative of three independent experiments. (d) Irradiated C57BL/6 mice were reconstituted with BM cells infected with control virus (white), wild-type (black), or mutant Nlrc4 (gray)-encoding virus. Serum IL-1β, IL-17A, and G-CSF were evaluated by ELISA 6 wk after BM transplantation. For all panels, the data shown are means ± SD (**, P < 0.01; n = 5). N.D.: not detected. The data in the figure are representative of two independent experiments.

Article Snippet: The concentration of G-CSF was measured with the Mouse G-CSF Quantikine ELISA kit (R&D Systems).

Techniques: Mutagenesis, Concentration Assay, Enzyme-linked Immunosorbent Assay, Infection, Control, Virus, Transplantation Assay, Irradiation

Journal: The Journal of Experimental Medicine

Article Title: An inherited mutation in NLRC4 causes autoinflammation in human and mice

doi: 10.1084/jem.20141091

Figure Lengend Snippet: Cold exposure of mu-Nlrc4 mice increases autoinflammation. (a) The footpads of C57BL/6 (wild type; white) and mu-Nlrc4 (black) mice were exposed to 4°C for 5 min. The thickness of each footpad was measured before and after exposure to the cold stimulus. The data are shown as the ratio of the thickness after exposure to the thickness before exposure. The data are shown as means ± SD (**, P < 0.01; n = 5). The data in the figure are representative of three independent experiments. (b) Irradiated C57BL/6 mice were reconstituted with BM cells infected with control virus (white), wild-type (black), or mutant Nlrc4 (gray)-encoding virus. The footpads were exposed to 4°C for 5 min. The thickness of each footpad was measured before and after exposure to the cold stimulus. The data are shown as the ratio of the thickness after exposure to the thickness before exposure. The data are shown as means ± SD (**, P < 0.01; n = 5). The data in the figure are representative of three independent experiments. (c) The entire bodies of control C57BL/6 and mu-Nlrc4 mice were exposed to 4°C for 1 min and kept for 3 min at room temperature, and then the serum concentrations of IL-1β were measured. The data are shown as means ± SD. N.D., not detected. (**, P < 0.01; n = 5). The data in the figure are representative of three independent experiments. (d) MC/9 cells infected with control retrovirus (white), retroviruses encoding wild-type (black), or mutant Nlrc4 (gray) were cultured at 32 or 37°C for 48 h. 293T cells were transfected with control vector (white), vectors encoding wild-type (black), or mutant NLRC4 (gray) and 48 h later, cells were washed and cultured at 32 or 37°C for 6 h. The levels of IL-1β in the supernatants were evaluated by ELISA. The data are shown as the mean ratio of the IL-1β level in cells cultured at 32°C to the level in cells cultured at 37°C. The data are shown as means ± SD (*, P < 0.05). The data in the figure are representative of three independent experiments. (e) Peripheral mononuclear cells from an FCAS patient or a healthy control were cultured at 32°C for 48 h in the presence (black) or absence (white) of a caspase inhibitor or cultured at 37°C for 48 h. The level of IL-1β in the supernatant was evaluated by ELISA. The data are shown as the mean ratio of the IL-1β level in cells cultured at 32°C to the level in cells cultured at 37°C. The data are shown as means ± SD (**, P < 0.01). The data in the figure are representative of two independent experiments.

Article Snippet: The concentration of G-CSF was measured with the Mouse G-CSF Quantikine ELISA kit (R&D Systems).

Techniques: Irradiation, Infection, Control, Virus, Mutagenesis, Cell Culture, Transfection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Experimental Medicine

Article Title: An inherited mutation in NLRC4 causes autoinflammation in human and mice

doi: 10.1084/jem.20141091

Figure Lengend Snippet: The accumulation of neutrophils in mu-Nlrc4 mice depends on IL-1β and IL-17A. (a) Spleen cells from C57BL/6 or mu-Nlrc4 mice at the age of 12 wk were stimulated with 25 ng/ml PMA and 1 µg/ml ionomycin in the presence of 2 µM monensin for 5 h and then stained with anti–TCR-β, anti–TCR-γδ, anti-B220, anti-CD11c, anti-NK1.1, anti-Gr1, and anti-CD11b antibodies. The cells were fixed and stained with anti–IL-17A antibody. The expression of IL-17A was analyzed by flow cytometry. The number in each rectangle indicates the percentage of IL-17A–positive cells in the lineage-negative fraction. The data in the figure are representative of five independent experiments. (b) mu-Nlrc4 mice at the age of 12 wk were injected with control IgG, anti-CD4, anti-CD8, anti-Thy1.2, anti-Gr1, or anti–IL-1β antibody five times at 3-d intervals. The serum IL-17A levels 2 d after final antibody treatment was measured by ELISA. Control sera from C57BL/6 (wild type) mice was used. The data shown are means ± SD (**, P < 0.01; n = 5 for all). The data in the figure are representative of three independent experiments. (c) mu-Nlrc4 mice at the age of 12 wk were injected twice with control rat IgG, anti-CD4, anti-CD8, or anti-Thy1.2 at 3 d intervals. The spleen cells from mice 1 d after the final injection were stained with anti-CD4 and anti–TCR-β (for anti-CD8–treated mice), anti-CD8 and anti–TCR-β (for anti-CD4–treated mice), or anti-CD4, anti-CD8, and anti–TCR-β antibodies (for anti-Thy1.2–treated mice). The data in the figure are representative of two independent experiments. (d) mu-Nlrc4 mice at the age of 12 wk were injected with control IgG, anti–IL-1β, anti–IL-17A, or anti–IL-1β, and anti–IL-17A antibody five times at 3-d intervals. The number of Gr1 + CD11b + cells in the spleen 2 d after the final antibody treatment was evaluated by flow cytometry. The data shown are means ± SD (*, P < 0.05; **, P < 0.01; n = 5 for all). The data in the figure are representative of three independent experiments.

Article Snippet: The concentration of G-CSF was measured with the Mouse G-CSF Quantikine ELISA kit (R&D Systems).

Techniques: Staining, Expressing, Flow Cytometry, Injection, Control, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: Spatiotemporally organized immunomodulatory response to SARS-CoV-2 virus in primary human broncho-alveolar epithelia

doi: 10.1016/j.isci.2023.107374

Figure Lengend Snippet:

Article Snippet: Anti-CSF3 AF594 (Clone CSF3/900) , Novus , Cat#NBP2-47934AF594 RRID: AB_2933966.

Techniques: Bacteria, Virus, Variant Assay, Recombinant, Red Blood Cell Lysis, Software, Membrane, Cell Culture

Journal: Cell stem cell

Article Title: Regulation of hematopoietic stem and progenitor cell mobilization by cholesterol efflux pathways

doi: 10.1016/j.stem.2012.04.024

Figure Lengend Snippet: Plasma IL-17 levels in WT and Abca1−/−Abcg1−/− recipient mice transplanted with either WT or Abca1−/−Abcg1−/− BM 8 weeks post-reconstitution (A). Plasma G-CSF levels in WT recipients transplanted with WT and Abca1−/−Abcg1−/− BM 14 weeks post-reconstitution and i.p injected with IgG control or 250µg IL-17 neutralizing antibody for 16h (B). Colony-forming unit numbers from the blood of these mice (C). Plasma IL-17 levels in WT and Abca1−/−Abcg1−/− mice treated with broad-spectrum antibiotics for 2 weeks to deplete commensal bacteria (D). IL-23 protein content in the spleen of WT and Abca1−/−Abcg1−/− recipient mice transplanted with either WT or Abca1−/−Abcg1−/− BM 8 weeks post-reconstitution (E). Plasma IL-17 levels in WT recipients transplanted with WT and Abca1−/−Abcg1−/− BM 14 weeks post-reconstitution and i.p injected with IgG control or 200µg IL-23R neutralizing antibody for 16h (F). Results are ± SEM of 5 to 6 animals per group. *P<0.05 vs. WT recipients transplanted with WT BM. #P<0.05 vs. IgG control. §P<0.05 vs. Abca1−/−Abcg1−/− recipients transplanted with Abca1−/−Abcg1−/− BM.

Article Snippet: For neutralizing antibody experiments, WT and Abca1 −/− Abcg1 −/− BM transplanted mice were i.p injected 16h before analysis with the following antibodies: anti-IL-3Rβ AF549, anti-Cxcl2 MAB452, anti-IL-23R MAB1686, anti-IL-17 MAB421 and anti-G-CSF MAB414 (all from from R&Dsystems).

Techniques: Injection

Journal: Cell stem cell

Article Title: Regulation of hematopoietic stem and progenitor cell mobilization by cholesterol efflux pathways

doi: 10.1016/j.stem.2012.04.024

Figure Lengend Snippet: Splenic IL-23 protein content (A), Colony-forming unit numbers (B), plasma G-CSF levels (C), and plasma IL-17 levels (D) in 28 weeks old Abca1fl/flAbcg1fl/fl (controls), LysM-Cre Abca1fl/flAbcg1fl/fl, and CD11c-Cre Abca1fl/flAbcg1fl/fl mice. Results are ± SEM of 5 to 6 animals per group. *P<0.05 vs. controls. Modulation of IL-23 mRNA expression levels in Abca1fl/flAbcg1fl/fl and LysM-Cre Abca1flfl-Abcg1fl/fl BM-derived macrophages (E) and CD11c-Cre Abca1fl/flAbcg1fl/fl BM-derived dendritic cells (F) after manipulation of plasma membrane cholesterol. Cells were incubated with 5mmol/L cyclodextrin (CD) for 30minutes before treatment with 50ng/mL lipopolysaccharide (LPS, TLR4 ligand) or 2,5µg/mL PolyI:C (TLR3 ligand) for 3 hours. IL-23 transcript levels were normalized to m36B4 mRNA amount. RNA levels were expressed as arbitrary units (a.u). Values are mean ± SEM of an experiment performed in quadruplicate. *P<0.05 vs. respective controls. §P<0.05 vs. conditions without cyclodextrin treatment.

Article Snippet: For neutralizing antibody experiments, WT and Abca1 −/− Abcg1 −/− BM transplanted mice were i.p injected 16h before analysis with the following antibodies: anti-IL-3Rβ AF549, anti-Cxcl2 MAB452, anti-IL-23R MAB1686, anti-IL-17 MAB421 and anti-G-CSF MAB414 (all from from R&Dsystems).

Techniques: Expressing, Derivative Assay, Incubation

Journal: Cell stem cell

Article Title: Regulation of hematopoietic stem and progenitor cell mobilization by cholesterol efflux pathways

doi: 10.1016/j.stem.2012.04.024

Figure Lengend Snippet: Quantification of BM macrophage subsets by flow cytometry in WT and Abca1−/−Abcg1−/− recipient mice transplanted with either WT or Abca1−/−Abcg1−/− BM 8 weeks post-reconstitution (A and B) and in WT recipients transplanted with WT and Abca1−/−Abcg1−/− BM 14 weeks post-reconstitution and i.p injected with IgG control or 250µg IL-17 neutralizing antibody for 16h (C and D). Results are ± SEM of 5 to 6 animals per group. *P<0.05 vs. WT. §P<0.05 vs. Abca1−/−Abcg1−/− recipients transplanted with Abca1−/−Abcg1−/− BM. Quantification of hematopoietic progenitors (E), monocytes and neutrophils (F) in WT BM cultures grown for 48h in liquid culture in presence of 10% of the indicated serum and 50ng/mL G-CSF neutralizing antibody (+) or control non-specific IgG (−). Results are ± SEM of 3 independent experiments. #P<0.05 vs. IgG control. †P<0.05 vs. 10% WT serum.

Article Snippet: For neutralizing antibody experiments, WT and Abca1 −/− Abcg1 −/− BM transplanted mice were i.p injected 16h before analysis with the following antibodies: anti-IL-3Rβ AF549, anti-Cxcl2 MAB452, anti-IL-23R MAB1686, anti-IL-17 MAB421 and anti-G-CSF MAB414 (all from from R&Dsystems).

Techniques: Flow Cytometry, Injection