Fig 2: E-selectin endothelium surface expression is differentially upregulated according to the vascular bed upon infection.a Flow cytometry analysis of cell surface expression of ICAM-1, VCAM-1, and E-selectin (CD62E) on HUVEC cells under resting conditions (Basal, open histograms) or following infection with Neisseria meningitidis for 2 h (Nm2h, red histograms) or 5 h (Nm5h, dark-red histograms) or an overnight incubation with 20 ng ml-1 TNFa gray histograms). Data are representatives of N = 3 independent experiments. The percentages of positive cells (above the dashed lines) are shown per condition and marker. b In vivo expression of E-selectin at the surface of different human vessel types (capillary, arteriole, and venule) following two (2 h p.i.) and four (4 h p.i.) hours of infection. Images (maximum intensity z-projection) are representative of N = 3 infected mice imaged independently. GFP-expressing Neisseria meningitidis appears in green and E-selectin in red following in vivo labeling by i.v. injection of PE-labeled anti-CD62E monoclonal antibody. Dashed lines delineate human vessels (UEA-1 lectin). Scale bar, 20 µm. c High-magnification view (yellow dashed square in panel b) of a bacterial microcolony at 4 h p.i. on the venular endothelium surface and the local upregulation of E-selectin expression. Scale bar, 5 µm. d Movies obtained from intravital imaging were used to quantify the numbers of neutrophils per square millimeter of venular endothelium during the first 4 h of the infection in presence of anti-E-selectin blocking antibody (red circles) or isotype control (white circles). Data are shown as the mean ± SEM. Quantifications were performed on n = 22 and 8 venules for E-selectin blocking and isotype control conditions, respectively, pooled from N = 3 infected mice imaged independently per condition. Two-tailed two-way ANOVA. e Representative image (maximum intensity z-projection) of venular colonization 4 h post infection by Neisseria meningitidis (green) in presence of the anti-E-selectin blocking antibody. The human endothelium was labeled using UEA-1 lectin (gray). The picture shown is representative of N = 3 mice imaged independently. Scale bar, 50 µm.

Fig 3: Functional analyses of hiPSCs-derived ECs (hiPSC-ECs) at day 12 of cultivation. (A) hiPSC-ECs formed tube-like structures when seeded on matrigel. Scale bars represent 200 µm. (B) Expression analysis of CD62E, ICAM-1, and VCAM-1 transcripts using qRT-PCR in unstimulated and TNF-α-stimulated hiPSC-ECs differentiated in vitronectin-coated 12-well plates. The mRNA levels were normalized to GAPDH mRNA levels, and the results are presented relative to the expression levels in hiPSCs. Results are shown as mean + SEM (n = 3). Statistical differences were determined using the paired t-test (** p < 0.01). (C) Flow cytometric analysis of CD62E-expressing hiPSC-ECs without and with 50 ng/mL of TNF-α stimulation differentiated in vitronectin-coated 12-well plates. Results are shown as mean + SEM (n = 3). Statistical differences were determined using paired t-test (** p < 0.01). (D) Representative immunocytochemistry images of hiPSC-ECs differentiated in vitronectin-coated 12-well plates without or with subsequent TNF-α stimulation and staining with a PE-labeled CD62E-specific antibody. Scale bars represent 100 µm. Samples were fixed, processed, and imaged identically. (E) Interaction of granulocytes (calcein AM: green) with unstimulated and TNF-α-stimulated hiPSC-ECs under static conditions. (F) Numbers of attached granulocytes to unstimulated and TNF-α-stimulated hiPSC-ECs under static conditions. Results are shown as mean + SEM (n = 3). Statistical differences were determined using the paired t-test (* p < 0.01; ** p < 0.01; *** p < 0.001).