Fig. 3: Histone-dependent binding of AGEs to macrophages.

a Binding of AGEs to mouse peritoneal cells. Left histogram, peritoneal cells were incubated alone (black line) or with Bt-BSA (blue line) or Bt-AGEs (red line), stained with streptavidin-APC, and analyzed by flow cytometry. Right two panels, Stained cells with CD11b and F4/80 were analyzed without or with gating on APC (+). b Cell-type specificity for AGEs binding. Peritoneal cells were incubated alone (NT) or with Bt-BSA (BSA) or Bt-AGEs (AGEs) and stained with streptavidin-BV421. Macrophages, Monocytes, Dendritic cells, Neutrophils, Eosinophils were gated and the bindings of Bt-BSA or Bt-AGEs were expressed as median fluorescent intensity (MFI) of BV421. Data are mean ± S.D. (n = 4, biologically independent experiments). Tukey–Kramer tests (two-sided), n.s. not significant (p > 0.05). c, d Binding of AGEs to J774A.1 (c) and RAW264.7 (d) cells. The cells were incubated alone (black line) or with either Bt-BSA (blue line) or Bt-AGEs (red line) and analyzed by flow cytometry. e Colocalization of H2B with AGEs. J774A.1 cells were treated with either Bt-BSA or Bt-AGEs and stained with anti-H2B antibody and streptavidin-alexa568. Scale bar, 10 µm. Representative image of three independent experiments. f Pull-down assay for the detection of H2B as a cell-surface AGEs-binding protein. J774A.1 cells were incubated with Bt-BSA or Bt-AGEs and then incubated with DTSSP. The detergent-resistant membrane fractions were subjected to pull-down, and the resulting precipitates were detected by western blotting using an anti-H2B antibody. Representative image of two independent experiments. g Inhibition of AGEs binding by anti-histones or anti-AGEs receptors antibodies. Cells were preincubated with antibody against histone H1, H2A, H2B, H3, H4, RAGE, AGER1, or CD36 prior to the treatment with Bt-AGEs. Relative AGEs bindings were determined by the MFI of APC. Data are mean ± S.D. (n = 4 for histone antibodies and n = 3 for known AGEs receptors antibodies, biologically independent experiments). Dunnett’s test (two-sided), relative to the control. h Effect of H2B overexpression on AGEs binding. J774A.1 cells were transfected with a bicistronic construct encoding H2B followed by IRES driving the translation of GFP (upper left panel) and the binding of Bt-AGEs was analyzed (n = 3, biologically independent experiments). Paired t test (two-sided).