Journal: Journal of autoimmunity

Article Title: Impaired Tip60-mediated Foxp3 acetylation attenuates regulatory T cell development in rheumatoid arthritis.

doi: 10.1016/j.jaut.2019.02.007

Figure Lengend Snippet: Fig. 3. Impaired Tip60 expression in RA patients. CD4+CD45RO−T cells from RA patients and controls were stimulated for 4 days, and (A–B) Tip60 gene expression was quantified by QPCR in naïve T cells from RA patients, HCs and OA patients. The results from 3 independent experiments involving 16 RA patients and 14 HCs are shown as scatter plots in relation to the donor age (A) and as bar graphs displaying the mean ± SEM of the RA patients, HCs and OA patients (B). (C) Tip60 insufficiency is independent of drug treatment (treated vs. treatment naïve), and Tip60 gene expression was measured in the naïve CD4+ T cells 4 days after TCR-mediated stimulation. (D) Tip60 transcript levels were correlated with the number of Treg cells in the PBMCs from RA patients. (E–F) Representative blotting for Tip60 and Foxp3 protein expression (E). The Tip60 and Foxp3 band intensities were adjusted to GAPDH. The western blotting experiment results analyzing 6 RA patients, 6 OA and 6 HCs are shown as the mean ± SEM (F). (G) Co-immunostaining of Foxp3 (green), acetylated-lysine (red), and Tip60 (white). Representative images are from one of three synovial tissue sections from the OA and RA patients. Bar, 20 μm. (H) Fluorescence intensities of Foxp3 (green), acetylated-lysine (red), acetylated-Foxp3 (combined green and red), and Tip60 (white) in the sections of synovial tissue from 6 OA and 6 RA patients are presented as the mean ± SEM. ***P < 0.001; n.s., non-significance. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Alternatively, Oligo duplex RNA specific for Tip60 and a control siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, USA).

Techniques: Expressing, Gene Expression, Western Blot, Immunostaining, Fluorescence

Journal: Journal of autoimmunity

Article Title: Impaired Tip60-mediated Foxp3 acetylation attenuates regulatory T cell development in rheumatoid arthritis.

doi: 10.1016/j.jaut.2019.02.007

Figure Lengend Snippet: Fig. 4. Foxp3 expression and Treg cell differentiation are regulated by Tip60. CD4+ T cells from healthy donors were transfected with control or Tip60-specific shRNA by nucleofection or treated with Tip60-specific inhibitor NU9056 (2 μM) at day 3 after bead stimulation. (A) The Tip60 transcript levels were quantified by QPCR 24 h after control and Tip60 siRNA transfection. (B) Representative blotting for Tip60 and Foxp3 expression. (C) Foxp3 acetylation was determined by a coimmunoprecipitation assay after transfection. (D) The Foxp3 levels in T cells were analyzed by flow cytometry in the GFP+ cell population after control and Tip60 shRNA transfection. Representative histograms for one control shRNA (blue) and one Tip60 shRNA (red); three independent experiments examining 6 HCs are presented as the mean ± SEM. (E–G) CD4+ T cells were transfected with the control or Tip60 siRNA and cultured under Treg- or Th17-polarizing conditions, and intracellular staining for Foxp3 and IL-17a was performed. (E) Representative dot plots for Treg and Th17 cell production. (F) Percentages of Tregs under Treg- polarizing conditions or CD4+Foxp3+ cells and CD4+IL-17a+ Th17 cells under Th17-polarizing conditions are presented as the mean ± SEM. (G) Suppression ability of the Tregs from control and Tip60 siRNA was analyzed by quantifying CFSE dilution. (H–M) Tip60 inhibitor: (H) Representative blotting for Foxp3 protein expression after NU9056 treatment. (I) Foxp3 acetylation was determined by a coimmunoprecipitation assay. (J) The Foxp3 protein levels in T cells were analyzed by flow cytometry. Representative histograms for DMSO (blue) and the Tip60 inhibitor (red); three independent experiments examining 6 HCs are presented as the mean ± SEM. (K–M) CD4+ T cells were treated with vehicle (DMSO) or NU9056 (2 μM) and cultured under Treg- or Th17-polarizing conditions. (K) Representative dot plots. (L) Percentages of Tregs or Th17 cells are presented as the mean ± SEM. (M) Suppression ability of the Tregs from vehicle and inhibitor treatment cells was analyzed by quantifying CFSE dilution. **P < 0.01; ***P < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Alternatively, Oligo duplex RNA specific for Tip60 and a control siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, USA).

Techniques: Expressing, Transfection, Control, shRNA, Co-Immunoprecipitation Assay, Cytometry, Cell Culture, Staining

Journal: Journal of autoimmunity

Article Title: Impaired Tip60-mediated Foxp3 acetylation attenuates regulatory T cell development in rheumatoid arthritis.

doi: 10.1016/j.jaut.2019.02.007

Figure Lengend Snippet: Fig. 5. Overexpression of Tip60 restores Treg cell differentiation. Naïve CD4+ T cells from RA patients were transduced with pEGFP-C2 or pEGFP-Tip60. The cells were transfected after anti-CD3/CD28 bead stimulation. (A) Representative blotting for Tip60 and Foxp3 protein expression after transfection. (B–C) The Foxp3 protein levels in naïve T cells were analyzed by flow cytometry in GFP+ cell population after transfection (B). Three independent experiments examining 6 RA patients are presented as the mean ± SEM (C). (D) Foxp3 acetylation was determined by a coimmunoprecipitation assay with anti-Foxp3, followed by im- munoblotting with an anti-acetylated-lysine antibody. Representative blots from three independent experiments are shown. (E–H) T cell differentiation: CD4+CD45RO−T cells were cultured under Treg- or Th17-polarizing conditions, re-stimulated with PMA/ionomycin and subjected to intracellular staining for Foxp3 and IL-17a. (E–F) Representative dot plots for Tregs (right) and Th17 cell (left) production. (G) Percentages of Tregs under Treg-polarizing condition or CD4+Foxp3+ cells and CD4+IL-17a+ Th17 cells under Th17-polarizing conditions from three independent experiments examining 6 RA patients are presented as the mean ± SEM. (H) Naïve CD4+ T cells from RA patients were transduced with pcDNA3.1and pcDNA3.1-Tip60. Suppression capability of the Tregs from cells transfected with control and Tip60 plasmids was analyzed by quantifying the CFSE dilution. The mean ± SEM of division indics are shown from 6 independent coculture experiments. **P < 0.01; ***P < 0.001.

Article Snippet: Alternatively, Oligo duplex RNA specific for Tip60 and a control siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, USA).

Techniques: Over Expression, Transduction, Transfection, Expressing, Cytometry, Co-Immunoprecipitation Assay, Cell Culture, Staining, Control

Journal: Journal of autoimmunity

Article Title: Impaired Tip60-mediated Foxp3 acetylation attenuates regulatory T cell development in rheumatoid arthritis.

doi: 10.1016/j.jaut.2019.02.007

Figure Lengend Snippet: Fig. 6. Tip60 controls the arthritogenic effect of T cells. Pairs of NSG mice were engrafted with synovial tissue from RA patients, CD45RO−PBMCs from healthy donors were transferred to the chimeric mice. The mice were divided into two groups, vehicle (DMSO) control and treatment with NU9056 group, where both treatments were delivered by intraperitoneal injection of 2 mg/kg/day for 9 days. (A) H&E staining was performed to compare the overall morphology of the synovium. Representative images of the synovium in the vehicle (n = 6) and Tip60 inhibitor-treated (n = 6) chimeric mice are shown. The data are representative of three independent experiments. (B) The intensity of synovial inflammation was compared by QPCR to assess TRB and TNFSF11 gene expression. All data are presented as the mean ± SEM from at least 6 different synovial grafts. (C–E) CD20 (C) and RANKL (D) were stained by immunohistochemistry in synovial tissue sections. The percentages of CD20+ B cells and RANKL-positive cells were determined by counting 9 different fields of synovial tissue sections and presented as the mean ± SEM (E). (F–H) T cell infiltration: (F) Representative images of anti-CD3 (red) and IL-17a staining (green) of the vehicle- (n = 6) and Tip60 inhibitor-treated (n = 6) chimeric mice. CD3+IL-17+ double positive cells are indicated by a yellow coloring. Bar, 20 μm. (G) Representative images of anti-Foxp3 staining (green) of the vehicle- (n = 6) and Tip60 inhibitor-treated (n = 6) samples. Bar, 20 μm. (H) The percentages of CD3+IL-17+ cells and Foxp3+ cells are presented as the mean ± SEM. (I) Co-immunostaining of Foxp3 (green), acetylated-lysine (red), and Tip60 (white). Representative images are from one of three synovial tissue from vehicle and Tip60 inhibitor-treated chimeric mice. Bar, 20 μm. (J) Fluorescence intensity of Foxp3, acetylated-Foxp3 (combined green and red) and Tip60 are presented as the mean ± SEM. (K) Expression of IL-6, TNF-α, IL-17, IL-10 and TGF-β in the synovium was measured by QPCR. The data are representative of three independent experiments. ***P < 0.001; n.s., non-significance. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Alternatively, Oligo duplex RNA specific for Tip60 and a control siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, USA).

Techniques: Control, Injection, Staining, Gene Expression, Immunohistochemistry, Immunostaining, Fluorescence, Expressing

Journal: Journal of autoimmunity

Article Title: Impaired Tip60-mediated Foxp3 acetylation attenuates regulatory T cell development in rheumatoid arthritis.

doi: 10.1016/j.jaut.2019.02.007

Figure Lengend Snippet: Fig. 7. Restoring Tip60 expression in RA T cells prevents arthritogenic effect. Pairs of NSG mice were engrafted with synovial tissue from RA patients and divided into two treatment groups. CD45RO−PBMCs from RA patients were transfected with either a control plasmid (pcDNA3.1) or pcDNA3.1-Tip60 and adop- tively transferred to the chimeric mice. (A) H&E staining to compare the overall morphology of the synovium from control and Tip60 plasmids transferred chimeric mice. Representative images of the synovium in the control (n = 6) and Tip60 plasmids (n = 6) samples are shown. Bar, 20 μm. (B) The intensity of synovial inflammation was compared by QPCR to assess TRB and TNFSF11 gene expression. The data are representative of three independent experiments. All data are presented as the mean ± SEM from at least 6 different synovial grafts. (C–E) CD20 (C) and RANKL (D) were stained by immunohistochemistry in synovial tissue sections from control and Tip60 plasmid-transfected chimeric mice. The percentages of CD20+ B cells and RANKL-positive cells are presented as the mean ± SEM (E). (F–H) T cell infiltration: (F) Representative images of anti-CD3 (red) and IL-17 staining (green) of the control (n = 6) and Tip60 plasmids (n = 6) samples. CD3+IL-17a+ cells are indicated with a yellow coloring. Bar, 20 μm. (G) Representative images of anti-Foxp3 staining (green) of the control (n = 6) and Tip60 plasmids (n = 6) samples. Bar, 20 μm. (H) The percentages of CD3+IL-17a+ cells and Foxp3+cells in randomly selected fields of synovial tissue sections are presented as the mean ± SEM. (I) Co-immunostaining of Foxp3 (green), acetylated-lysine (red), and Tip60 (white). Representative images are from one of three synovial tissue from control and Tip60 vector transfected mice. Bar, 20 μm. (J) Fluorescence intensity of Foxp3, acetylated-Foxp3 (combined green and red) and Tip60 are presented as the mean ± SEM. (K) Expression of IL-6, TNF-α, IL-17, IL-10 and TGF-β in the synovium was measured by QPCR. The data are representative of three independent experiments. **P < 0.01; ***P < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Alternatively, Oligo duplex RNA specific for Tip60 and a control siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, USA).

Techniques: Expressing, Transfection, Control, Plasmid Preparation, Staining, Gene Expression, Immunohistochemistry, Immunostaining, Fluorescence