Journal:

Article Title: Type-I IFN Signaling Suppresses an Excessive IFN-? Response and Thus Prevents Lung Damage and Chronic Inflammation During Pneumocystis (PC) Clearance in CD4 T Cell-Competent Mice

doi: 10.2353/ajpath.2010.091158

Figure Lengend Snippet: Gene and conditioning tree of differentially regulated genes expressed in pulmonary CD11c+ cells from IFNAR−/− and wild-type mice as determined by gene array analysis. Gene array analysis was performed with RNA from pulmonary CD11c+ cells isolated from wild-type and IFNAR−/− mice at day seven and 14 post PC infection. RNA from three individual animals per group and time point was assessed. Displayed is a gene and conditioning tree of significantly regulated genes between the comparison groups that demonstrates high reproducibility.

Article Snippet: CD11c + cells were isolated from the lungs of three individual PC-infected IFNAR −/− and wild-type mice at day 7 and 14 post infection using CD11c (N418) magnetic microbeads from Miltenyi, Auburn, CA (MACS).

Techniques: Isolation, Infection, Comparison

Journal:

Article Title: Type-I IFN Signaling Suppresses an Excessive IFN-? Response and Thus Prevents Lung Damage and Chronic Inflammation During Pneumocystis (PC) Clearance in CD4 T Cell-Competent Mice

doi: 10.2353/ajpath.2010.091158

Figure Lengend Snippet: Gene expression analysis of pulmonary CD11c+ cells from IFNAR−/− and wild-type mice after PC lung infections reveal a unique inflammatory profile between the groups. Demonstrated are the expression profiles of some of the regulated genes identified between the comparison groups. Expression intensity is displayed as normalized ratio to allow for multiple chip comparison. A: Expression level of genes coding for cell surface markers characterizing the analyzed cell population. B: Expression level of various type I IFN-regulated genes between the groups. C: Expression of various SOCS (suppressor of cytokine signaling molecules) between the groups. D: Differential expression level of various cytokines and growth factors. E: Expression of various chemokines. All genes displayed in B–E are significantly regulated between the groups with a P value <0.02 as identified through a two-way analysis of variance using a Student t test and Benjamini and Hochberg multiple test correction.

Article Snippet: CD11c + cells were isolated from the lungs of three individual PC-infected IFNAR −/− and wild-type mice at day 7 and 14 post infection using CD11c (N418) magnetic microbeads from Miltenyi, Auburn, CA (MACS).

Techniques: Gene Expression, Expressing, Comparison, Quantitative Proteomics

Journal:

Article Title: Type-I IFN Signaling Suppresses an Excessive IFN-? Response and Thus Prevents Lung Damage and Chronic Inflammation During Pneumocystis (PC) Clearance in CD4 T Cell-Competent Mice

doi: 10.2353/ajpath.2010.091158

Figure Lengend Snippet: Quantitative PCR confirms reduced SOCS1 expression in pulmonary CD11c+ cells isolated from IFNAR−/− versus wild-type mice at day seven post Pneumocystis infection. Relative quantification of SOCS1 expression in pulmonary CD11c+ cells isolated from three individual wild-type and IFNAR−/− mice was performed using TaqMan technique with gene-specific probes and primers for mouse SOCS1 as the target gene and GAPDH as the endogenous control gene. Expression levels are demonstrated in Log10 relative quantification with the expression level in wild-type mice as reference. Statistical differences were assessed between the two comparison groups using a Student t test and are indicated with *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: CD11c + cells were isolated from the lungs of three individual PC-infected IFNAR −/− and wild-type mice at day 7 and 14 post infection using CD11c (N418) magnetic microbeads from Miltenyi, Auburn, CA (MACS).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Isolation, Infection, Quantitative Proteomics, Control, Gene Expression, Comparison

Journal: The Journal of Experimental Medicine

Article Title: Epithelial and dendritic cells in the thymic medulla promote CD4 + Foxp3 + regulatory T cell development via the CD27–CD70 pathway

doi: 10.1084/jem.20112061

Figure Lengend Snippet: CD70 on CD8α + thymic cDCs contributes to T reg cell development. (A) Thymic sections were stained sequentially using anti-CD70 mAb, Alexa Fluor 568–conjugated anti-IgG, FITC-conjugated anti-CD11c mAb, and APC-conjugated anti-CD8α mAb. Arrowheads indicate cells with double staining for CD70 and CD11c. (B and C) DCs were enriched from WT or Cd70 Cre/Cre thymi and analyzed by flow cytometry. (B) Shown are representative plots of CD8α versus SIRPα expression on gated CD11c + B220 lo DCs. (C) CD8α + and SIRPα + DC subsets were purified from WT and Cd70 Cre/Cre thymi, RNA was isolated, and RT-PCR was performed on cDNA. Shown are PCR products from cDNA samples amplified with primers specific for CD70 and HPRT. (D) CD8α + and SIRPα + CD11c + B220 low/− cDCs and CD11c + B220 + pDCs were purified from the pooled thymi of two WT or Cd70 Cre/Cre mice. The purified DC subsets were co-cultured for 5 d with purified CD4 + CD25 − WT thymocytes. After culture, gated CD4 + cells were analyzed for expression of Foxp3 and CD25. Shown is one representative plot of Foxp3 and CD25 expression on gated CD4 + cells in the presence of the indicated DC population. (E) Data from D, expressed as the percentage of Foxp3 + CD25 + cells among gated CD4 + cells in the presence of the indicated DC populations. Results are from four to five separate wells over two independent experiments. The Mann–Whitney U rank sum test was used to analyze results (*, P < 0.05). Error bars are SEM.

Article Snippet: Mouse thymus was harvested and roughly diced into small pieces using scissors in 25 ml IMDM containing 1 mg/ml collagenase type IV (Worthington) and 25 μg/ml DNase (Sigma-Aldrich) and incubated at 37°C for 1 h. Liberated cells were filtered through 100-μM nylon mesh and incubated with CD11c microbeads (Miltenyi Biotech) for 30 min before being run through a MACS column (Miltenyi Biotech).

Techniques: Staining, Double Staining, Flow Cytometry, Expressing, Purification, Isolation, Reverse Transcription Polymerase Chain Reaction, Amplification, Cell Culture, MANN-WHITNEY

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

Article Title: β-Catenin Signaling Drives Differentiation and Proinflammatory Function of IRF8-Dependent Dendritic Cells

doi: 10.4049/jimmunol.1402453

Figure Lengend Snippet: β-catenin stabilization directs splenic DC progenitors towards CD8α+ DC development. (A) Intracellular β-catenin expression in naïve Ex3fl/fl and Ex3DC−/− splenic CD11c+ cells. (B) Intracellular β-catenin levels in splenic CD4+ T cells isolated from Ex3fl/fl and Ex3DC−/− mice. The data show results from an individual mouse that is representative of of at least 3 experiments with 3–5 mice per group. (C) Western blot analysis of β-catenin in bone marrow-derived DC from Ex3fl/fl and Ex3DC−/− mice following cytoplasmic (C) and nuclear (N) fractionation. Antibodies against PARP and Rab5 were used for nuclear and cytoplasmic loading controls, respectively. The data are from 1 independent trial. (D and E) Comparison pre-cDC populations from the (D) bone marrow and (E) spleen of Ex3fl/fl and Ex3DC−/− mice by flow cytometry. Numbers in representative plots represent percentages of relevant populations within the indicated gate. Bar graphs show mean percentages plus standard error (S.E.) of relevant populations. The data represent the combination of 2 independent experiments (n=10 mice per group). (F) Levels of splenic pre-CD8α+ DC, defined as CD11c+CD8α−B220−CD24+, in Ex3fl/fl and Ex3DC−/− mice by flow cytometry. The data are representative of 3 independent experiments, each involving 4–5 mice per group. **, p<0.01; ***, p<0.001.

Article Snippet: A single round of positive selection using CD11c + magnetic bead sorting was performed for purification of total splenic DC from single cell suspensions (Stem Cell Technologies), while two-step magnetic bead sorting, with an initial negative selection to enrich for DC followed by CD8α + positive selection (Miltenyi Biotec), was performed to isolate CD8α + splenic DC.

Techniques: Expressing, Isolation, Western Blot, Derivative Assay, Fractionation, Comparison, Flow Cytometry

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

Article Title: β-Catenin Signaling Drives Differentiation and Proinflammatory Function of IRF8-Dependent Dendritic Cells

doi: 10.4049/jimmunol.1402453

Figure Lengend Snippet: β-catenin stabilization expands splenic CD8α+ and plasmacytoid DC populations. (A–G) Mature DC subset analysis of naïve Ex3fl/fl and Ex3DC−/− splenocytes by flow cytometry. (A and B) Percentage and total number of CD11c+ cells in Ex3fl/fl and Ex3DC−/− spleens. (C–E) Percentage and total number of (C and D) CD8α+ DC and (C and E) CD11b+ DC in naïve Ex3fl/fl and Ex3DC−/− spleens. The data are representative of at least 3 independent experiments (n=3–5 mice per group). (F and G) Percentage and total number of B220+PDCA-1+ plasmacytoid DC in naïve Ex3fl/fl and Ex3DC−/− spleens. (H and I) Mature DC subset analysis of naïve Ex3fl/fl and Ex3DC−/− lung tissue by flow cytometry. (H) Plots from representative mice and (I) percentage of CD103+CD11b− lung DC for multiple mice are shown. (J–L) Mature DC subset analysis of naïve Ex3fl/fl and Ex3DC−/− intestinal lamina propria by flow cytometry. (J) Plots from representative mice and percentages of (K) CD103+CD11b− and (L) CD103+CD11b+ intestinal DC for multiple mice are shown. Dots in relevant graphs represent results from individual mice. Bar graphs display means and standard errors of individual mice. The data are representative of at least 2 independent experiments (n=3–5 mice per group). *, p<0.05; **, p<0.01; ***, p<0.001.

Article Snippet: A single round of positive selection using CD11c + magnetic bead sorting was performed for purification of total splenic DC from single cell suspensions (Stem Cell Technologies), while two-step magnetic bead sorting, with an initial negative selection to enrich for DC followed by CD8α + positive selection (Miltenyi Biotec), was performed to isolate CD8α + splenic DC.

Techniques: Flow Cytometry

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

Article Title: β-Catenin Signaling Drives Differentiation and Proinflammatory Function of IRF8-Dependent Dendritic Cells

doi: 10.4049/jimmunol.1402453

Figure Lengend Snippet: β-catenin signaling controls Irf8 expression. (A) Semi-quantitative PCR analysis of Nfil3, Batf3, Id2, and Irf8 mRNA in CD11c+ splenocytes magnetically purified from naïve Ex3fl/fl and Ex3DC−/− mice. mRNA levels were normalized to GAPDH. The data are representative of 2 independent experiments (n=2–3 mice per group) (B) Representative flow cytometric plots of IRF8 expression by Ex3fl/fl and Ex3DC−/− CD8α− and CD8α+ splenic DC. (C) MFI of IRF8 within Ex3fl/fl and Ex3DC−/− CD8α− and CD8α+ DC and (D) the percent of CD8α+ DC expressing IRF8 are shown. Dots represent results from individual mice. The data are the combined results of 2 experiments, and the experiment was independently performed at least 3 times (n=4–5 mice per group). (E) Representative FACS plot of IRF4 expression and IRF4 MFI in Ex3fl/fl and Ex3DC−/− CD11c+ splenocytes. The data are representative of 3 independent experiments (n=4 mice per group). (F) Chromatin immunoprecipitation of naïve Ex3DC−/− Flt3L DC cultures with control IgG or β-catenin antibody followed by quantitative PCR to determine Irf8 promoter occupancy. DNA levels were normalized to 1% input chromatin. The data are representative of 2 independent experiments. (G) Quantitative PCR analysis of Axin2 and Irf8 gene expression in BMDC following 5 hr culture with DMSO or ICG-001. Fold change is relative to DMSO control. The data are from one independent trial. (H and I) Intracellular expression of IRF8 and β-catenin following ICG-001 treatment of BMDC (H) or MutuDC1940 cells (I). The data are representative of 2 (MutuDC1940 cells) and 4 (BMDC) independent experiments with 3 replicates per treatment per experiment. *, p<0.05; **, p<0.01; ***, p<0.001.

Article Snippet: A single round of positive selection using CD11c + magnetic bead sorting was performed for purification of total splenic DC from single cell suspensions (Stem Cell Technologies), while two-step magnetic bead sorting, with an initial negative selection to enrich for DC followed by CD8α + positive selection (Miltenyi Biotec), was performed to isolate CD8α + splenic DC.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Purification, Chromatin Immunoprecipitation, Control, Gene Expression

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

Article Title: β-Catenin Signaling Drives Differentiation and Proinflammatory Function of IRF8-Dependent Dendritic Cells

doi: 10.4049/jimmunol.1402453

Figure Lengend Snippet: β-catenin stabilization enhances IL-12 production by CD8α+ DC. (A) IL-12p40 production by naïve Ex3fl/fl and Ex3DC−/− splenocytes stimulated in vitro with LPS, STAg, or media control measured by ELISA. (B) IL-12p40 production by splenic CD11c+ DC magnetically purified from Ex3fl/fl and Ex3DC−/− mice stimulated in vitro with LPS, STAg, or media control measured by ELISA. (C) IL-12p40 production by CD8α+ and CD8α− DC DC purified from naïve Ex3fl/fl and Ex3DC−/− splenocytes following in vitro stimulation with media or STAg for 48 hr measured by ELISA. (D) IL-12p40 secretion by Ex3fl/fl splenocytes pre-treated with ICG-001 for 5 hr and then stimulated overnight with LPS or STAg measured by ELISA. (E) IL-12p40 production by splenocytes (106) from Ex3DC−/− mice cultured for 5 hr with 5 μM ICG-001 or DMSO and then stimulated with media, LPS (100 ng/ml), or STAg (25 μg/ml) overnight. (F) IL-12p40 production by MutuDC1940 cells (105) pre-treated with 20 μM ICG-001 or DMSO for 2 hr and then stimulated with media or STAg (25 μg/ml) overnight. The data are representative of at least 3 (A, F) and 2 (B–E) independent experiments, each involving 3–5 mice per group, except (C), which used pooled samples from 3 mice per experiment. *, p<0.05; **, p<0.01; ***, p<0.001.

Article Snippet: A single round of positive selection using CD11c + magnetic bead sorting was performed for purification of total splenic DC from single cell suspensions (Stem Cell Technologies), while two-step magnetic bead sorting, with an initial negative selection to enrich for DC followed by CD8α + positive selection (Miltenyi Biotec), was performed to isolate CD8α + splenic DC.

Techniques: In Vitro, Control, Enzyme-linked Immunosorbent Assay, Purification, Cell Culture

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

Article Title: β-Catenin Signaling Drives Differentiation and Proinflammatory Function of IRF8-Dependent Dendritic Cells

doi: 10.4049/jimmunol.1402453

Figure Lengend Snippet: Constitutive DC β-catenin signaling increases the proinflammatory cytokine response to Toxoplasma. (A) Survival of Ex3fl/fl and Ex3DC−/− mice following i.p. infection with Toxoplasma Type II strain ME49 (25 cysts) (n=4–6 mice per group). The data are representative of at least 3 experiments. (B) Quantitative PCR amplification of parasite (B1 gene) and host DNA (ASL gene) isolated from Ex3fl/fl and Ex3DC−/− spleens 9 days post-infection. Parasite load is displayed as a ratio of parasite genomes to host genomes (n=3–4 mice per group). (C) IL-12p40 production by CD11c+ DC magnetically separated from Day-6 post-infection Ex3fl/fl and Ex3DC−/− splenocytes and cultured overnight without additional stimulation (n=3 mice per group). (D) IL-12p70 production by bulk splenocytes from Day-6 post-infection Ex3fl/fl and Ex3DC−/− mice (n=3 mice per group). The data are representative of 2 independent experiments. (E) IL-12p40 and IFN-γ production by splenocytes from Day-10 post-infection Ex3fl/fl and Ex3DC−/− mice cultured for 72 hr without additional stimulation (n=3–5 mice per group). The data are representative of 3 independent experiments. (F) IL-12p40, IFN-γ, and TNF-α levels in serum collected from Day-9 post-infection Ex3fl/fl and Ex3DC−/− mice (n=3–5 mice per group). The data are representative of 2 independent experiments. The means and S.E. of individual mice are shown. *, p<0.05; **, p<0.01.

Article Snippet: A single round of positive selection using CD11c + magnetic bead sorting was performed for purification of total splenic DC from single cell suspensions (Stem Cell Technologies), while two-step magnetic bead sorting, with an initial negative selection to enrich for DC followed by CD8α + positive selection (Miltenyi Biotec), was performed to isolate CD8α + splenic DC.

Techniques: Infection, Real-time Polymerase Chain Reaction, Amplification, Isolation, Cell Culture