Journal: The Journal of Biological Chemistry

Article Title: Human Memory, but Not Naive, CD4 + T Cells Expressing Transcription Factor T-bet Might Drive Rapid Cytokine Production *

doi: 10.1074/jbc.M114.608745

Figure Lengend Snippet: Kinetics of cytokine secretion by activated naive and memory CD4+ T cells. Naive and memory CD4+ T cells were isolated from CD4+ T cells by MACS microbeads, and the purity of naive or memory CD4+ T cells was more than 97% as determined by flow cytometry (a). Naive and memory CD4+ T cells were stimulated with (open histogram) or without (shaded histogram) PMA and ionomycin in the presence of BFA for 6 h. Cells were stained by intracellular staining and analyzed by FACS (b). Purified naive and memory CD4+ T cells were stimulated with immobilized anti-CD3 (1 μg/ml) and anti-CD28 (1 μg/ml) for 0 to 72 h, and the concentrations of cytokines IFN-γ, IL-2, and TNF-α were detected by ELISA (c). Data are representative of three separate experiments with similar results.

Article Snippet: Human ELISA kits for cytokines IFN-γ, IL-2, and TNF-α were purchased from BD Biosciences.

Techniques: Isolation, Flow Cytometry, Staining, Purification, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: Virulence from vesicles: Novel mechanisms of host cell injury by Escherichia coli O104:H4 outbreak strain

doi: 10.1038/srep13252

Figure Lengend Snippet: ( a ) Caco-2, HCT-8, and HT-29 cells were incubated with OMVs LB226692 (containing 58 ng/ml of Stx2a, 80 ng/ml of flagellin, and 950 ng/ml of LPS) or OMVs C227-11Φcu (containing 80 ng/ml of flagellin and 950 ng/ml of LPS) for 15 min, 1 h, 3 h, and 24 h; IL-8 in cell culture supernatants was quantified by a Single-Analyte IL-8 ELISArray; * P < 0.05 (unpaired Student’s t test) for comparison between OMVs LB226692 and C227-11Φcu. ( b ) Cells were exposed for 24 h to: OMVs or isolated H4 flagellin (80 ng/ml) in the absence of antibodies; OMVs or flagellin which had been preincubated with anti-H4 antibody; OMVs or flagellin after cell preincubation with anti-TLR5 antibody; OMVs or flagellin which had been preincubated with anti-H4 antibody after cell preincubation with anti-TLR5 antibody. IL-8 secretion was quantified as above; ** P < 0.01 or *** P < 0.001 (one-way ANOVA) for comparison between each OMV preparation or flagellin without and after preincubation with each respective antibody combination. ( c ) IL-8 secretion was quantified in cells incubated for 24 h with OMVs or isolated O104 LPS (950 ng/ml) without polymyxin B (polyB-) or with OMVs or LPS which had been preincubated with polymyxin B (polyB+); * P < 0.05 (one-way ANOVA) for comparison between each OMV preparation or LPS without and after polymyxin B preincubation; ** P < 0.01 (one-way ANOVA) for comparison between each OMV preparation and isolated O104 LPS. ( d ) Cells were incubated for 24 h with 10-fold dilutions of OMVs LB226692 or C227-11Φcu containing the indicated amounts of flagellin and LPS; IL-8 secretion was quantified as above. Data in panels ( a – d ) are means ± standard deviations from three independent experiments. ( e ) Detection of TLR5, TLR4 and MD-2 in lysates of Caco-2, HCT-8, and HT-29 cells using immunoblot. Signals were visualised with Chemi Doc XRS imager. Sizes of immunoreactive bands are shown on the right side. Crops of representative immunoblots are shown. Full immunoblots are shown in .

Article Snippet: IL-8 production elicited by OMVs, H4 flagellin, and O104 LPS was quantified with the Single-Analyte IL-8 ELISArray (Qiagen).

Techniques: Incubation, Cell Culture, Comparison, Isolation, Western Blot

Journal: Frontiers in Immunology

Article Title: NR4A Receptors Differentially Regulate NF-κB Signaling in Myeloid Cells

doi: 10.3389/fimmu.2017.00007

Figure Lengend Snippet: NR4As positively regulate TLR4 driven MIP-3α in human and murine monocyte/macrophage cells . (A) Undifferentiated THP-1 cells transduced with shRNA directed against scrambled non-target control (Sc), NR4A2 (A2), or NR4A3 (A3) were treated with 1 µg/ml lipopolysaccharide (LPS) for 8 h, followed by media collection. (B) Undifferentiated THP-1 cells transduced with shRNA directed against scrambled non-target control, NR4A2, or NR4A3 were treated with 1 µg/ml LPS for 0, 2, and 8 h. (C) Human primary PBMCs were exposed to 1 µg/ml LPS for 2 h. (D) Undifferentiated THP-1 cells were pretreated with 10 µM NF-κB inhibitor (NF-κBi) for 1 h followed by the addition of 1 µg/ml LPS for 2 h. (E,F) Mouse embryonic fibroblast (MEF) cells lacking p65 −/− and wild-type (WT) controls were exposed to 1 µg/ml LPS for 4 h (E) and 2 h (F) . (G) Murine raw mac 264.7 cells transduced with shRNA directed against scrambled non-target control (pretreated with 10 µM NF-κB inhibitor (NF-κBi) for 1 h) and NR4A2 were subsequently treated with of 1 µg/ml LPS for 1 h. (H) MEF cells lacking p65 −/− and WT controls were exposed to 1 µg/ml LPS for 2 h. (I,J) Undifferentiated THP-1 cells were pretreated with 100 nM Cytosporone-B (Csn-b) for 30 min followed by the addition of 100 ng/ml LPS for a further 1.5 h. Analysis: ELISA was performed at time indicated for MIP-3α protein (A) . RNA was isolated and RT-PCR was performed at indicated times to assess levels of MIP-3α, NR4A2, NR4A3, TNFα, and control gene GAPDH (B–D,F,H,I,J) . Whole-cell lysates were prepared at indicated time followed by Western blot analysis performed for both p65 and loading control β-tubulin (E) . Densitometric analysis included for Western blot data was determined using LI-COR ® Image Studio Lite version 3.1 and band density of target protein (p65) normalized to loading control (β-Tubulin) are displayed above relevant treatments. Un, untreated control. Data are expressed as fold over untreated control (FOC) or pg/ml ± SEM for n = minimum of three individual experiments. ** p < 0.01, *** p < 0.001 treatments compared to untreated control (Un). # p < 0.05, ## p < 0.01, ### p < 0.001 treatments compared displayed here using a bar attachment.

Article Snippet: MCP-1 and MIP-3α (RayBiotech) protein levels were measured using an enzyme-linked immunosorbent assay.

Techniques: Transduction, shRNA, Enzyme-linked Immunosorbent Assay, Isolation, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Frontiers in Immunology

Article Title: NR4A Receptors Differentially Regulate NF-κB Signaling in Myeloid Cells

doi: 10.3389/fimmu.2017.00007

Figure Lengend Snippet: NR4A2 does not require DNA binding in order to regulate TLR4-driven MIP-3α . (A) Graphical representation [showing ≈500 bp upstream and ≈200 downstream of the transcription start site (TSS) (TSS is indicated on graphic as a black arrowhead)] of the NR4A (shown as yellow box) and NF-κB (shown as purple box) DNA-binding motifs (NBRE and NRE, respectively) on the MIP-3α human and murine promoter analyzed using the Genomatix software suite program MatInspector for 1,000 bp upstream of the TSS. Expanded out regions marked by a blue lined box is ≈200 bp upstream of TSS showing the exact sequence of the NR4A and NF-κB-binding motifs. Human and murine promoter are shown aligned using the ApE plasmid editor software, red boxes indicate gaps, and red boxes with a # indicate a mismatch. (B) Mouse embryonic fibroblast (MEF) cells were transfected with 500 ng of wild-type (WT) p-CMX-NR4A2, p-CMX-NR4A2 mutant (C283G), and backbone control plasmid (BB). Following 48 h incubation, cells were treated with 1 µg/ml lipopolysaccharide (LPS) for 2 h. (C) MEF cells lacking p65 −/− and WT controls were exposed to 1 µg/ml LPS for 2 h. Analysis: RNA was isolated and subsequent qRT-PCR performed to assess levels of MIP-3, IL-6, and control gene β-actin (B,C) . Data are expressed as fold over untreated control (FOC) ± SEM for n = minimum of 2 (B) and 6 (C) individual experiments. * p < 0.05, ** p < 0.01 treatments compared to untreated control (Un). # p < 0.05 treatments compared displayed here using a bar attachment.

Article Snippet: MCP-1 and MIP-3α (RayBiotech) protein levels were measured using an enzyme-linked immunosorbent assay.

Techniques: Binding Assay, Software, Sequencing, Plasmid Preparation, Transfection, Mutagenesis, Incubation, Isolation, Quantitative RT-PCR

Journal: Frontiers in Immunology

Article Title: NR4A Receptors Differentially Regulate NF-κB Signaling in Myeloid Cells

doi: 10.3389/fimmu.2017.00007

Figure Lengend Snippet: NR4As are regulators of Relb expression during TLR4 stimulation . (A) Mouse embryonic fibroblast (MEF) cells lacking p65 −/− and wild-type (WT) control were exposed to 1 µg/ml lipopolysaccharide (LPS) for 2 h. (B) MEF cells lacking p65 −/− , Relb −/− , and WT control were exposed to 1 µg/ml LPS for 4 h. (C) MEF cells lacking Relb −/− and WT control were exposed to 1 µg/ml LPS for 2 h. (D) Undifferentiated THP-1 cells were pretreated with 10 µM NF-κB inhibitor (NF-κBi) for 1 h followed by the addition of 1 µg/ml LPS for 2 h. (E) Human primary PBMCs were exposed to 1 µg/ml LPS for 2 h. (F) Undifferentiated THP-1 cells transduced with shRNA directed against scrambled non-target control, NR4A2, or NR4A3 were treated with 1 µg/ml LPS for 2 h. Analysis: RNA was isolated and RT-PCR was performed at indicated times to assess levels of MIP-3α, Relb, and control gene GAPDH (A,C–F) . Whole-cell lysates were prepared at indicated time followed by Western blot analysis performed for both Relb and loading control β-tubulin (B) . Densitometric analysis included for Western blot data was determined using LI-COR ® Image Studio Lite version 3.1, and band density of target protein (Relb) normalized to loading control (β-Tubulin) are displayed above relevant treatments. Un, untreated control. Data are expressed as fold over untreated control (FOC) ± SEM for n = minimum of three individual experiments. ** p < 0.01, *** p < 0.001 treatments compared to untreated control (Un). # p < 0.05, ### p < 0.001 treatments compared displayed here using a bar attachment.

Article Snippet: MCP-1 and MIP-3α (RayBiotech) protein levels were measured using an enzyme-linked immunosorbent assay.

Techniques: Expressing, Transduction, shRNA, Isolation, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Frontiers in Immunology

Article Title: NR4A Receptors Differentially Regulate NF-κB Signaling in Myeloid Cells

doi: 10.3389/fimmu.2017.00007

Figure Lengend Snippet: Summary diagram . TLR4 or TNFα receptor stimulation increases the activity of NF-κB and the expression of its target genes NR4A1–3 (NR4A), MCP-1, MIP-3α, and Relb. Loss of NR4A potentiates this induction of MCP-1, while reducing the induction of Relb and MIP-3α. Furthermore using mouse embryonic cells, we identify MIP-3α is a novel Relb target gene. Therefore, NR4A receptors act as repressors of inflammatory-NF-κB-driven MCP-1 (indicated by a red T bar) and enhancers of NF-κB-driven Relb and subsequently MIP-3α. Using a NR4A2 DNA-binding mutant reveals that NR4A2 does not require the ability to bind DNA in order to enhance/repress target genes simultaneously.

Article Snippet: MCP-1 and MIP-3α (RayBiotech) protein levels were measured using an enzyme-linked immunosorbent assay.

Techniques: Activity Assay, Expressing, Binding Assay, Mutagenesis

Journal: Oncotarget

Article Title: Atorvastatin inhibits the immediate-early response gene EGR1 and improves the functional pro of CD4 + T-lymphocytes in acute coronary syndromes

doi: 10.18632/oncotarget.15420

Figure Lengend Snippet: + CD25 high T-cells. Experimental conditions are reported in Figure . A . The percentage of CD4 + CD25 high Tcells producing IL-10 increased significantly after treatment with atorvastatin (P for trend < 0.001). Data are presented as median and 95% CI. * P = 0.034 untreated cells vs 3μg/mL of atorvastatin; † P = 0.022 untreated cells vs 10μg/mL of atorvastatin; ‡ P < 0.001 untreated cells vs 26μg/mL of atorvastatin. B . The mean fluorescence intensity (MFI) of intracellular IL-10 expression by CD4 + CD25 high T-cells also increased significantly after atorvastatin treatment (P for trend < 0.001). Data are presented as mean±SD. * P = 0.056 untreated cells vs 10μg/mL of atorvastatin; † P < 0.001 untreated cells vs 26μg/mL of atorvastatin. C . IL-10 was measured by high-sensitivity ELISA in aliquots of 1mL of whole blood incubated for 24 hours without and with increasing doses of atorvastatin: 3-10-26μg/ml. IL-10 concentrations significantly increased after atorvastatin treatment (P for trend = 0.024). * P = 0.025 untreated cells vs 3μg/mL of atorvastatin; † P = 0.016 untreated cells vs 10μg/mL of atorvastatin; ‡ P = 0.058 untreated cells vs 26ug/mL of atorvastatin.

Article Snippet: Plasma levels of IL-10 and IFN-γ were measured with high-sensitivity ELISA kits (human-IL-10, Aushon Biosystems, Billerica, MA; human-IFN-γ, Bender MedSystem, Vienna, Austria), according to the manufacturer's instructions.

Techniques: Fluorescence, Expressing, Enzyme-linked Immunosorbent Assay, Incubation