Fig. 4 Brd4 binds and activates the BLIMP1 promoter
(A ) Effect of Brd4 inhibition on the expression of BLIMP1 in human CD19+ B cells. Human CD19+ B cells were cultured with anti-IgM, rCD40L, CpG ODN2006, rIL-10 and rIL-15 in the presence or absence of PFI-1 or JQ1 for 7 days. BLIMP1 expression was measured by qRT-PCR. Data are expressed as mean ± SEM from 5 independent experiments. (B ) Effect of Brd4 deficiency on expression of BLIMP1 in CD19+ B cells from Brd4-CKO mice. Isolated splenic B cells were stimulated with LPS (10 μg/mL) and IL-4 (10 ng/mL) for 48 h. Data are expressed as mean ± SEM of 6 independent experiments. (C ) HEK-293T cells were cotransfected with BLIMP1 reporter and Brd4 siRNA or control siRNA. Luciferase activity was measured 48 hours post-transfection. (D ) The potential Brd4 binding sites were evaluated by ChIP assay using anti-Brd4 or anti-IgG antibodies in B220+B cells from spleen of wild type (FL/FL) mice. The presence of Brd4 binding site on BLIMP1 promotor was measured by qPCR. *P <0.05, **P <0.01 versus control (ctrl) or wild type (FL/FL) or siC,#P <0.05 versus co-stim.
Fig. 5 The effects of Brd4 inhibition on B cell differentiation and Ig production in SLE. (A and B ) Purified CD19+ B cells from SLE patients (n=18) were cultured with anti-IgM, rCD40L, CpG ODN2006, rIL-4 and rIL-2 as indicated in the presence of Brd4 inhibitor PFI-1. The generation of naïve B cells (CD19+IgD+CD27-), memory B cells (CD19+IgD-CD27+), plasmablasts (CD19+IgD-CD27++) and plasma cells (CD19+CD38++CD138+) was evaluated after 7 days in culture. The cells were stained for various surface markers and analyzed by flow cytometry. The numbers in the quadrants represent the percentage of cells in culture (A ). Data are mean ± SEM (B ). (C and D) The effects of Brd4 inhibitor on differentiation of plasmablasts and plasma cells from SLE patients (n=5). Purified CD19+ B cells were cultured with anti-IgM (5μg/ml), rCD40L (1μg/ml), CpG ODN2006 (5μM), rIL-10 (50ng/ml) and rIL-15 (10ng/ml). The percentage of plasmablasts and plasma cells were examined after 5 days in culture by flow cytometry. Data are mean ± SEM from five independent experiments (D ). (E and F ) Effect of Brd4 inhibition on apoptosis (E ) and proliferation (F ) of activated lupus CD19+ B cells. Purified B cells were cultured with no stimulus (nil), co-stimulation (anti-IgM, rCD40L, CpG ODN2006, rIL-4 and rIL-2) in the presence of PFI-1 for 7 days. For detection of apoptosis, cells were washed and stained with annexin V and PI and analyzed by flow cytometry. For measurement of proliferation, cells were analyzed for CFSE dilution. (G ) IgG and IgM production detected by ELISA. The results are the mean ± SEM from 8 independent experiments. *P <0.05 vernus no stim,#P <0.05 vernus co-stim without PFI-1
Fig 6 Effect of Brd4 inhibitor PFI-1 on hypergammaglobulinemia and nephritis in MRL/lpr mice MRL/lpr mice (12 weeks of age) were treated with vehicle (DMSO) or 2 mg/kg of PFI-1 for 6 weeks (n=8 mice per group). (A ) Effect of PFI-1 on s erum levels of total IgG, IgG1, IgG2a and IgM. Serum samples obtained from 6-month-old MRL/lpr mice were used as the standard control. (B and C ) Effect of PFI-1 on glomerulonephritis of MRL/lpr mice. Representative kidney sections stained with PAS (B) and frozen sections stained for IgG and C3 deposition (C). Original magnification × 40. (D) Urinary protein levels in mice at 12, 14, 16 and 18 weeks of age. (E) Serum creatinine (Cr) and blood urea nitrogen (BUN) levels at the end of the experiment. Bars in A, D and E show the mean ± SEM. *P<0.05 vs DMSO. (F and G ) Effect of Brd4 inhibitor PFI-1 on the percentages of plasma cells from spleen, lymph node and peripheral blood mononuclear cells (PBMC) of MRL/lpr mice. The cell surface markers of B cell subsets were analyzed by flow cytometry. The numbers are the percentages of cells within the quadrants. The bars represent the mean ± SEM. *P <0.05 vs DMSO. (H ) Effect of PFI-1 on the BLIMP1 expression of splenic B cell from MRL/lpr mice. BLIMP1 expression was determined by quantitative RT-PCR. Data represent the mean ± SEM from 8 mice. *P <0.05, **P<0.01 vs DMSO. (I-K ) Effect of Brd4 deficiency on nephritis and percentages of plasma cells in Brd4-CKO mice with pristane-induced lupus model. (I) Urinary protein levels in FL/FL mice (FL/FL, n=6), pristane–injected CKO mice (CKO-SLE, n=6), and pristane–injected FL/FL mice (FL/FL-SLE, n=6) at 0, 3 and 6 months after pristane injection. (J ) Representative kidney sections from CKO and FL/FL mice stained with hematoxylin and eosin (H&E) for analysis of glomerular size and mesangial expansion. (K ) Effect of Brd4 deficiency on frequency of plasma cell in mice with pristane-induced lupus model. The frequency of plasma cells from spleen (SP), lymph node (LN) and peripheral blood mononuclear cells (PBMC) was evaluated using flow cytometric analysis. The numbers are the percentages of cells within the quadrants. The bars represent the mean ± SEM. *P < 0.05 versus FL/FL-SLE.