Fig 3. DENV NS1-induced HPA-1 secretion leading to glycocalyx degradation and hyperpermeability is MIF dependent.

(A) HUVECs were treated with PBS or 20 μg/ml NS1 recombinant proteins for the indicated time, followed by collection of the supernatants for CD138 detection by ELISA. (n = 4) (B) HUVECs were treated with PBS, 20 μg/ml NS1, or 20 μg/ml NS1 mixed with 10 μg/ml anti-NS1 antibodies (2E8 or DN5C6). After 24 h of incubation, the culture medium was collected, and the concentration of CD138 was determined by ELISA. (n = 3) (C) HUVECs seeded as monolayers in upper Transwell chambers were treated with PBS, 20 μg/ml NS1 or 20 μg/ml NS1 mixed with DMSO or the indicated concentration of OGT 2115. After 24 h, endothelial permeability was determined by a Transwell permeability assay, as described in the Materials and Methods section. (n = 3) (D) HUVECs were treated with PBS or 20 μg/ml NS1 with or without 5 μM OGT 2115. After 24 h, the cell culture medium was collected, and the CD138 concentration was measured by ELISA. (n = 5) (E) HUVECs were treated with PBS or 20 μg/ml NS1 with or without 100 μM p425, 50 μM ISO-1, or 10 μg/ml anti-MIF antibodies, as indicated. After 24 h, the cell culture medium was collected, and the HPA-1 concentration was measured by ELISA. (n = 3) (F) HUVECs were treated with PBS or 20 μg/ml NS1 with or without p425, ISO-1 or anti-MIF antibodies. After 24 h, the cell culture medium was collected, and the CD138 concentration was measured by ELISA. (n = 5) (G) HUVECs were treated with PBS or NS1 (20 μg/ml) with or without anti-MIF polyclonal antibodies (10 μg/ml) for 24 h. The distribution of HPA-1 (red) and CD138 (green) was assessed by staining with specific antibodies. Sialic acid expression on HUVECs monolayers was assessed by staining with WGA-FITC (green). *P<0.05, **P<0.01, ***P<0.001; ns, not significant; unpaired t-test (panel A), Kruskal-Wallis ANOVA (panel B, C, D, E and F).