Fig. 4.

Relative TEER of human endothelial cells treated with acute YF human serum samples and effects of YFV NS1 treatment on endothelial glycocalyx. (a) Confluent monolayers of human endothelial cells cultured in Transwell inserts were treated or not with 10% serum from three different groups: Severe YF (n = 24, missing data for 15 samples); Non-severe YF (n = 10, missing data for 8 samples); Controls (n = 11): healthy individuals. YFV NS1 (10 μg/mL) was used as positive control. The transendothelial electrical resistance (TEER) was measured from 2 to 10 h post-treatment. Graph shows mean ± SD of relative TEER for each treatment group. (b) Area under the curve (AUC) of negative peaks for the effects of the different treatments on TEER. AUC values represent the sum of the trapezoidal areas (taking into account only the negative peaks) under the curve formed by the data points. Median values of AUC for each group were compared using Kruskal–Wallis’ test. Error bars represent interquartile range (IQR). Outliers were defined according to Tukey’s method, which identifies outliers using the interquartile range (IQR; i.e., the difference between the first and third quartiles [Q3 - Q1]) as any data point below the first quartile (25th percentile) minus 1.5 times the IQR (Q1 - 1.5∗IQR) or above the third quartile (75th percentile) plus 1.5 times the IQR (Q3 + 1.5∗IQR). (c, d) Human endothelial cell monolayers grown on gelatin-coated coverslips were treated with medium only or 10 μg/mL of YFV NS1 for 3 h. After fixation, cell surface syndecan-1 was stained in red and nuclei in blue (Hoechst). (c) Representative figures of cell monolayers stained for syndecan-1 in red and nuclei in blue. (d) Quantification of syndecan-1 protein on the cells surface expressed as mean fluorescence intensity (MFI). NS1 treatment was compared to medium-only treatment by unpaired t-test. Error bars present SD of the mean. (e, f, g) Human endothelial cell monolayers grown on gelatin-coated coverslips were treated with medium only or with different concentrations of YFV NS1 for 6 h. (e) Quantification of syndecan-1 in cell supernatants by ELISA. An in-house-produced mouse anti-YFV NS1 polyserum with or without 2.5 μg/mL of YFV NS1 was used as a control. Treatments were compared with medium-only treatment by one-way ANOVA + Dunnett’s test. Error bars present SD of the mean. (f) Representative figures of cell monolayers stained for sialic acid in red and nuclei in blue. (g) Quantification of sialic acid on the cell surface expressed as MFI. NS1 treatments were compared to medium-only treatment by one-way ANOVA + Dunnett’s multiple comparison test. Error bars present SD of the mean. Significant p-values are shown in the graphs. Acute YF disease was defined by detection of viral RNA by RT-qPCR in serum. Samples were run in duplicate for the TEER assays. IFA and ELISA data are the results of three independent experiments (n = 3).