File S1 - Role of Serine Proteases in the Regulation of Interleukin-877 during the Development of Bronchopulmonary Dysplasia in Preterm Ventilated Infants

Supporting information. Figure S1, Chemokines in preterm BALF. Peak concentration of (a) CXCL1, (b) CXCL2 and (c) CXCL5 in preterm BALF. Groups are represented on the x-axis while concentration of chemokines (pg/ml) are represented on the y-axis. Each point represents peak value from a single infant and bars are at medians (* = p77 ELISA. Antigens IL-877 (open circles) and IL-872 (open squares) were captured by (a) N11 and (b) BD capture antibody. Both were detected by the BD detection antibody. Concentration of the antigens is represented on the x-axis (pg/ml) while absorbance at 450 nm (corrected at 570 nm) is represented on the y-axis. Points represent means (± SEM) of three independent experiments. Values were compared by two-way ANOVA with Boferroni's post-test (*** = p77. Concentration of peak total IL-8 (pg/ml) and corresponding concentration of IL-877 (pg/ml) are plotted on the x- and y-axis respectively. Each point represents a single infant and correlation was tested by calculating Spearman's coefficient. Figure S4, Gestation and IL-877 expression. Correlation of gestation at birth with (a) concentration of IL-877 and (b) percentage of IL-8 expressed as IL-877, in preterm BALF. Birth gestation of infants (weeks) is plotted on the x-axis while first day IL-877 concentration (pg/ml) or proportion of total IL-8 (percentage) in preterm BALF is plotted on the y-axis. Each point represents a single infant and correlation was tested by calculating Spearman's coefficient. Figure S5, Expression of IL-877 from cells. Concentration (pg/ml) of total IL-8 (open bars) and IL-877 (shaded bars) from (a) airway epithelial cells and term cord-blood (b) PMNs & (c) monocytes, both unstimulated (U) and when stimulated (S) with IL-1β (airway epithelial cells) or LPS (cord-blood cells). Cell-lines and incubation conditions are represented on the x-axis while concentration (pg/ml) is on the y-axis. Bars are at means (± SEM) of at least three separate experiments. Figure S6, Panel of controls. Concentration of IL-877 detected by ELISA at 0 hours and after incubation in different conditions for 18 hours. Conditions are detailed on the x-axis and concentration of IL-877 (pg/ml) on the y-axis. Open bars represent buffer controls, grey bar represent BALF samples at 0 hour and black bar represents BALF samples after 18 hours incubation (n = 18). All bars are at means (± SEM). Conditions were compared by one-way ANOVA with Dunnets post-hoc test comparing against a control column (BALF at 0 hour) (*** = p77 by purified human neutrophil (a) elastase, (b) cathepsin-G and (c) proteinase 3 at varying concentration. Ratio of enzyme: substrate is represented on the x-axis while the fold-change of total IL-8 (circles) and IL-877 (squares) from original concentration is represented on the y-axis. Points plotted are means (± SEM) of three independent experiments. Concentration of IL-877 is compared to corresponding concentration of total IL-8 by two-way ANOVA with Bonferroni's post-test (* = p72 (grey bars) and rhIL-877 (black bars). Details of conditions are represented on the x-axis while expression of MMP-9 is represented on the y-axis (expressed as a fold change compared to the positive control). Bars are at means (± SEM) of three independent experiments. Difference in means was compared by one-way ANOVA with Tukey's post-test (comparing all pairs of columns). */*** = significant compared to negative control; ## = significant compared to rhIL-872 at a concentration of 10−8 M (* = p77 by (a) buffer, (b) purified elastase and, (c) purified cathepsin G showing concentration of recovered total IL-8 (squares), IL-877 (circles) and MMP-9 from degranulation of neutrophils (triangles) by the products of conversion. Time (hours) is represented on the x-axis while IL-8/MMP-9 (fold change from 0-hour values) is represented on the y-axis. Points plotted are means (± SEM) of three independent experiments. Statistical differences in concentration compared to buffer-control at each time-point was tested by 2-way ANOVA with Bonferroni's post-test. (*** = p72 by purified proteinase-3. Recovery of total IL-8 (expressed as a fold change from 0-hour) on incubation with buffer (circles) and proteinase-3 (squares) over 24 hours. Time (hours) is represented on the x-axis while fold-change in concentration (compared to concentration at “0-hour”) is represented on the y-axis. Points plotted are means (± SEM) of three independent experiments. Differences in concentration was tested by two-way ANOVA with Bonferroni's post-test (*** = p (DOC)