Journal: Arthritis & rheumatology (Hoboken, N.J.)
Article Title: EZH2 modulates the DNA methylome and controls T cell adhesion through junctional adhesion molecule-A in lupus patients
Figure Lengend Snippet: Effect of EZH2 on lymphocyte adhesion. (A) EZH2-overexpressing stimulated CD4+ T cells showed increased adhesion to HMVECs compared to cells transfected with an empty vector (n=8, control vs. EZH2 vector 1.0 ± 0.0 vs. 1.94 ± 0.74, p=0.02). (B) The increased adhesion of EZH2-overexpressing CD4+ T cells was blocked by neutralizing antibodies to JAM-A (One-way ANOVA p=0.002; n=6, control vector: IgG vs. JAM-A 1.00 ± 0.09 vs. 0.92 ± 0.15, post-hoc p=0.99; EZH2 vector: IgG vs. JAM-A 1.77 ± 0.57 vs. 1.04 ± 0.38, post-hoc p=0.01. Control vector IgG group vs. EZH2 vector IgG group post-hoc p=0.008). (C) Naïve CD4+ T cells from lupus patients stimulated overnight adhere to HMVECs significantly more than cells from healthy controls (n=6, control vs. SLE 1.0 ± 0.0 vs. 2.43 ± 2.31, p=0.002). (D) Blocking JAM-A reduced the ability of lupus CD4+ T cells to adhere to HMVECs (One-way ANOVA p=0.007; n=6, control: IgG vs. JAM-A 1.00 ± 0.09 vs. 1.05 ± 0.38, p=1.00; SLE: IgG vs. JAM-A 2.19 ± 1.08 vs. 1.12 ± 0.31, p=0.03. Control IgG group vs. SLE IgG group p=0.01). (E) Treating stimulated lupus CD4+ T cells with EZH2 inhibitor DZNep reduced EZH2 mRNA levels significantly (n=5, PBS vs. DZNep 0.013 ± 0.005 vs. 0.005 ± 0.002, p=0.02]. (F) At the protein levels, DZNep decreased EZH2, JAM-A, H3K27me3 levels in stimulated lupus CD4+ T cells. (G) Pre-treating stimulated CD4+ T cells from lupus patients with DZNep for 3 days significantly reduced their adhesion to HMVECs compared to cells treated with PBS (n=6, PBS vs. DZNep: 1.0 ± 0.0 vs. 0.42 ± 0.21, p=0.002). (H) Representative pictures of the adhesion assay: stimulated CD4+ T cells (in green) isolated from a lupus patients showed increased ability to adhere to HMVECs (in red) compared to a healthy control, and DZNep significantly reduced adhesion of CD4+ T cells from the same lupus patient. (I) Pre-treating stimulated CD4+ T cells isolated from lupus patients with GSK126 for 3 days led to significant reduction in cell adhesion compared to cells treated with DMSO (n=6, DMSO vs. GSK126: 1.0 ± 0.0 vs. 0.47 ± 0.32, p=0.048). Results are expressed as mean +/− SD and p<0.05 was considered significant.
Article Snippet: Stimulated EZH2-overexpressing CD4+ T cells (50,000 cells/well in 100 μl RPMI medium) were labeled with Calcein AM (cell-permeant dye, 5 μM; Invitrogen) in the presence or absence of neutralizing antibodies to JAM-A (50 μg/ml, BioLegend) or isotype control (Mouse IgG1, κ, BioLegend).
Techniques: Transfection, Plasmid Preparation, Blocking Assay, Cell Adhesion Assay, Isolation