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

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Interleukin-17 Enhancement of the Interleukin-6 Signaling Cascade in Astrocytes

doi: 10.4049/jimmunol.1000142

Figure Lengend Snippet: A, IL-17RA and IL-17RC mRNA expression was determined by RT-PCR in RAW264.7 cells (positive control), and primary astrocytes, in duplicate. B, Primary astrocytes were treated with medium (UN), IL-6/R (IL-6, 10 ng/ml and sIL-6R, 25 ng/ml), IL-17 (25 ng/ml) or IL-6/R plus IL-17 for up to 24 h, and levels of IL-6 and GAPDH mRNA expression were determined by RT-PCR and QRT-PCR. C, Primary astrocytes were treated with medium (UN), IL-6/R, different concentrations of IL-17 (1-50 ng/ml) or IL-6/R + IL-17 (1-50 ng/ml) for 4 h, and levels of IL-6 mRNA expression were determined by QRT-PCR. D, Primary astrocytes were treated with medium (UN), IL-6, sIL-6R or IL-17 in various combinations for 4 h, and levels of IL-6 mRNA expression were determined by QRT-PCR. E, Primary astrocytes were treated with medium (UN), IL-6/R, IL-17 or IL-6/R plus IL-17 for 2 - 48 h, and supernatants were analyzed for IL-6 protein using ELISA. Experiments shown are representative of at least three experiments. All data are the mean ± SD of three experiments. *, p ≤0.05; **, p ≤0.01.

Article Snippet: Recombinant human IL-6, recombinant human soluble IL-6 Receptor (sIL-6R) and recombinant mouse IL-17 were from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Positive Control, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Interleukin-17 Enhancement of the Interleukin-6 Signaling Cascade in Astrocytes

doi: 10.4049/jimmunol.1000142

Figure Lengend Snippet: A, Astrocytes were incubated with medium (UN), IL-6/R, IL-17 or IL-6/R plus IL-17 for 15 and 30 min, and then cell lysates were immunoblotted with antibodies against IκBα and GAPDH. The basal level of the untreated sample was set at 100, and the percentage change of IκBα upon IL-6/R, IL-17 or IL-6/R + IL-17 treatment compared with the basal value. B, Astrocytes were incubated with medium (UN), IL-6/R, IL-17 or IL-6/R plus IL-17 for 30 and 60 min, and then cell lysates were immunoblotted with antibodies against phospho-p65 Ser 536, p65 and GAPDH. C, Astrocytes were incubated with medium (UN), IL-6/R, IL-17 or IL-6/R plus IL-17 for 15 and 30 min, and then cell lysates were immunoblotted with antibodies against phospho-ERK1/2, ERK1/2 and GAPDH. D, Astrocytes were incubated with medium (UN), IL-6/R, IL-17 or IL-6/R and IL-17 for 30 min and 60 min, and then cell lysates were immunoblotted with antibodies against phospho-p38, p38 and GAPDH. E, Astrocytes were incubated with medium (UN), IL-6/R, IL-17 or IL-6/R plus IL-17 for 30 and 60 min, and then cell lysates were immunoblotted with antibodies against phospho-JNK, JNK and GAPDH. The basal level of the untreated sample was set at 1.0 and fold activation upon IL-6/R, IL-17 or IL-6/R + IL-17 treatment was compared with that value (B, C, D and E). Representative of at least three experiments.

Article Snippet: Recombinant human IL-6, recombinant human soluble IL-6 Receptor (sIL-6R) and recombinant mouse IL-17 were from R&D Systems (Minneapolis, MN).

Techniques: Incubation, Activation Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Interleukin-17 Enhancement of the Interleukin-6 Signaling Cascade in Astrocytes

doi: 10.4049/jimmunol.1000142

Figure Lengend Snippet: A, Astrocytes were cultured in absence or presence of IL-17 followed by IL-6/R treatment for 4 h. Actinomycin D (5 ng/ml) was then added, and cells harvested at 0, 30, 60, 120, and 240 min after addition. The abundance of IL-6 mRNA was determined by QRT-PCR. B, C, DMSO vehicle, BAY 11 (5 μM), U0126 (10 μM), SB203580 (10 μM) or JNKi II (10 μM) were added to cultures 1 h before cytokine addition, and then astrocytes were incubated with medium, IL-6/R, IL-17 or IL-6/R plus IL-17 for 4 h. Levels of IL-6 mRNA expression were determined by QRT-PCR. All data are the mean ± SD of three experiments. **, p ≤ 0.01; NS = not significant.

Article Snippet: Recombinant human IL-6, recombinant human soluble IL-6 Receptor (sIL-6R) and recombinant mouse IL-17 were from R&D Systems (Minneapolis, MN).

Techniques: Cell Culture, Quantitative RT-PCR, Incubation, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Interleukin-17 Enhancement of the Interleukin-6 Signaling Cascade in Astrocytes

doi: 10.4049/jimmunol.1000142

Figure Lengend Snippet: A, Primary astrocytes were treated with medium (UN), IL-6/R, IL-17 or IL-6/R plus IL-17 for 90 min, and then cells were cross-linked with formaldehyde. Soluble chromatin was subjected to immunoprecipitation with Abs against p65, P-p65, c-Fos, c-Jun, or normal rabbit IgG. PCR analysis of the positive control (input) indicates that soluble chromatin samples obtained from each time point had equal amounts of chromatin fragments containing the IL-6 promoter. B, Primary astrocytes were treated as above. Soluble chromatin was subjected to immunoprecipitation with Abs against histone acetylation (Ac-H3 and Ac-H4), p300, CBP, RNA Pol II or normal rabbit IgG. The basal level of the untreated sample was set at 1.0 and fold activation upon IL-6/R, IL-17 or IL-6/R plus IL-17 treatment was compared with that value. Representative of at least three experiments.

Article Snippet: Recombinant human IL-6, recombinant human soluble IL-6 Receptor (sIL-6R) and recombinant mouse IL-17 were from R&D Systems (Minneapolis, MN).

Techniques: Immunoprecipitation, Positive Control, Activation Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Interleukin-17 Enhancement of the Interleukin-6 Signaling Cascade in Astrocytes

doi: 10.4049/jimmunol.1000142

Figure Lengend Snippet: A, Astrocytes were treated with medium (UN), IL-6/R, IL-17 or IL-6/R plus IL-17 for 2 - 8 h, and levels of SOCS3 mRNA expression were determined by QRT-PCR. B and C, Astrocytes were transfected with SOCS3 siRNA (100 nM) or siRNA control (100 nM) for 48 h. Transfected cells were then treated with medium (UN) or IL-6/R plus IL-17 for 4 h. Levels of SOCS3 (B) and IL-6 (C) mRNA expression was determined by QRT-PCR. D, Astrocytes were transfected with SOCS3 siRNA or siRNA control for 48 h. Transfected cells were then treated with medium (UN) or IL-6/R plus IL-17 for 24 h, and supernatants analyzed for IL-6 protein by ELISA. E, SOCS3 floxed astrocytes were infected with GFP as control or GFP-Cre for deletion. After 48 h in culture, cells were harvested. Digestion of genomic DNA distinguishes the full-length (fl) and excised alleles (Δ). F, SOCS3 floxed astrocytes were infected with GFP as control or GFP-Cre for deletion. After 48 h in culture, the cells were treated with medium (UN), IL-6/R, IL-17 or IL-6/R plus IL-17 for 4 h, and levels of IL-6 mRNA expression determined by QRT-PCR. G, SOCS3 floxed astrocytes were infected with GFP as control or GFP-Cre for deletion. After 48 h in culture, cells were incubated with medium (UN), IL-6/R, IL-17 or IL-6/R plus IL-17 for 16 or 24 h, and supernatants analyzed for IL-6 protein by ELISA. All data are the mean ± SD of three experiments. *, p ≤ 0.05;**, p ≤ 0.01.

Article Snippet: Recombinant human IL-6, recombinant human soluble IL-6 Receptor (sIL-6R) and recombinant mouse IL-17 were from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Quantitative RT-PCR, Transfection, Enzyme-linked Immunosorbent Assay, Infection, Incubation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Interleukin-17 Enhancement of the Interleukin-6 Signaling Cascade in Astrocytes

doi: 10.4049/jimmunol.1000142

Figure Lengend Snippet: To evaluate NF-κB and MAPK activation in the absence or presence of SOCS3, SOCS3 floxed astrocytes were infected with GFP as control or GFP-Cre for deletion. After 48 h in culture, cells were treated with medium (UN) or IL-6/R plus IL-17 for 15, 30, 60 or 120 min, and then cell lysates were immunoblotted with antibodies against phospho-p65 Ser 536, p65, phospho-p38, p38, phospho-JNK, JNK or GAPDH. The basal level of the untreated GFP infected sample was set at 1.0 and fold activation upon IL-6/R plus IL-17 treatment compared with that value. Representative of at least three experiments.

Article Snippet: Recombinant human IL-6, recombinant human soluble IL-6 Receptor (sIL-6R) and recombinant mouse IL-17 were from R&D Systems (Minneapolis, MN).

Techniques: Activation Assay, Infection

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Interleukin-17 Enhancement of the Interleukin-6 Signaling Cascade in Astrocytes

doi: 10.4049/jimmunol.1000142

Figure Lengend Snippet: A, IL-6/R and IL-17 activate the NF-κB and MAPK pathway, which then induce IL-6 gene expression. The synergistic effect of these two mediators depends on NF-κB, p38 and JNK MAPK activity. Activated NF-κB p65, c-Fos and c-Jun bind to the IL-6 promoter. Concurrent with NF-κB and MAPK recruitment, IL-6/R and IL-17 leads to the recruitment of coactivators CBP and p300, modifications in AcH3 and AcH4, and recruitment of RNA Pol II to the IL-6 promoter, which results in transcriptional activation of the IL-6 gene. IL-17 can also enhance IL-6/R induced SOCS3 expression, and SOCS3 inhibits IL-6/R plus IL-17-induced NF-κB and MAPK activation, which results in a reduction of IL-6 gene expression in astrocytes. B, Naive CD4+ T cells, after activation by signaling through the T cell receptor and co-stimulatory molecules, can differentiate into Th17 cells in the presence of IL-6, TGF-β, IL-1 and IL-23. IL-17 together with IL-6/R triggers a positive-feedforward loop of IL-6 expression in astrocytes, which may also influence Th17 cell differentiation. SOCS3 participates in these processes as a negative feedback regulator. See text for details.

Article Snippet: Recombinant human IL-6, recombinant human soluble IL-6 Receptor (sIL-6R) and recombinant mouse IL-17 were from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Activity Assay, Activation Assay, Cell Differentiation