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 (* = p<0.05). Figure S2, Modification of IL-877 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 (*** = p<0.001). Figure S3, Expression of IL-8 and IL-877. 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) (*** = p<0.001). Figure S7, Protease dose-response. Conversion of IL-877 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 (* = p<0.05, ** = p<0.01, *** = p<0.001). Figure S8, Establishing the neutrophil degranulation assay. MMP-9 expressed from neutrophils after degranulation by control conditions (open bars), rhIL-872 (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 (* = p<0.05, *** = p<0.001, ## = p<0.01). Figure S9, Functional activity of IL-8 isoforms. Summary of processing of rhIL-877 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. (*** = p<0.001). Figure S10, Processing of rhIL-872 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<0.001). (DOC)补充资料。补充图S1：早产患儿支气管肺泡灌洗液（bronchoalveolar lavage fluid, BALF）中的趋化因子。(a) CXCL1、(b) CXCL2及(c) CXCL5在早产BALF中的峰值浓度。横轴为分组，纵轴为趋化因子浓度（pg/ml）。每个数据点代表1名婴儿的峰值浓度，柱形代表中位数（* = p<0.05）。补充图S2：IL-877酶联免疫吸附测定（enzyme-linked immunosorbent assay, ELISA）的方法优化。抗原IL-877（空心圆圈）与IL-872（空心方块）分别由(a) N11及(b) BD捕获抗体进行捕获，二者均通过BD检测抗体完成标记。横轴为抗原浓度（pg/ml），纵轴为450 nm处吸光度（经570 nm校正）。数据点为3次独立实验的平均值（±标准误（standard error of the mean, SEM））。组间差异采用双因素方差分析（two-way ANOVA）结合邦费罗尼事后检验（Bonferroni's post-test）进行比较（*** = p<0.001）。补充图S3：IL-8与IL-877的表达水平。总IL-8峰值浓度（pg/ml）与对应IL-877浓度（pg/ml）分别绘制于横轴与纵轴。每个数据点代表1名婴儿，相关性通过计算斯皮尔曼相关系数（Spearman's correlation coefficient）进行检验。补充图S4：胎龄与IL-877的表达关联。早产患儿出生胎龄与(a) BALF中IL-877浓度及(b) 以IL-877形式表达的IL-8占总IL-8比例的相关性。横轴为婴儿出生胎龄（周），纵轴为首次检测的早产BALF中IL-877浓度（pg/ml）或总IL-8的占比（百分比）。每个数据点代表1名婴儿，相关性通过计算斯皮尔曼相关系数进行检验。补充图S5：细胞来源的IL-877表达。(a) 气道上皮细胞及(b) 足月脐带血中性粒细胞（polymorphonuclear neutrophils, PMNs）与(c) 单核细胞中总IL-8（空心柱）及IL-877（阴影柱）的浓度（pg/ml），分别设置未刺激组（U）与刺激组（S）：气道上皮细胞采用IL-1β刺激，脐带血细胞采用脂多糖（lipopolysaccharide, LPS）刺激。横轴为细胞系及培养条件，纵轴为浓度（pg/ml）。柱形代表至少3次独立实验的平均值（±SEM）。补充图S6：对照组合集。ELISA在0小时及经不同条件孵育18小时后检测到的IL-877浓度。横轴为孵育条件，纵轴为IL-877浓度（pg/ml）。空心柱为缓冲液对照，灰色柱为0小时的BALF样本，黑色柱为孵育18小时后的BALF样本（n=18）。所有柱形代表平均值（±SEM）。组间差异采用单因素方差分析（one-way ANOVA）结合邓尼特事后检验（Dunnett's post-hoc test），以0小时BALF样本为对照进行比较（*** = p<0.001）。补充图S7：蛋白酶剂量反应效应。不同浓度的纯化人中性粒细胞(a) 弹性蛋白酶、(b) 组织蛋白酶G及(c) 蛋白酶3对IL-877的转化作用。横轴为酶与底物的比例，纵轴为总IL-8（圆圈）与IL-877（方块）相对于初始浓度的倍数变化。数据点为3次独立实验的平均值（±SEM）。IL-877浓度与对应总IL-8浓度的差异采用双因素方差分析结合邦费罗尼事后检验进行比较（* = p<0.05, ** = p<0.01, *** = p<0.001）。补充图S8：中性粒细胞脱颗粒实验的建立。经对照条件、重组人IL-872（recombinant human IL-872, rhIL-872，灰色柱）及重组人IL-877（recombinant human IL-877, rhIL-877，黑色柱）诱导脱颗粒后，中性粒细胞表达的基质金属蛋白酶9（matrix metalloproteinase 9, MMP-9）水平。横轴为实验条件详情，纵轴为MMP-9表达水平（以阳性对照的倍数变化表示）。柱形代表3次独立实验的平均值（±SEM）。组间均值差异采用单因素方差分析结合图基事后检验（Tukey's post-test）（对所有柱形组进行两两比较）。*/*** = 与阴性对照相比具有显著性差异；## = 与浓度为10^−8 M的rhIL-872相比具有显著性差异（* = p<0.05, *** = p<0.001, ## = p<0.01）。补充图S9：IL-8亚型的功能活性。(a) 缓冲液、(b) 纯化弹性蛋白酶及(c) 纯化组织蛋白酶G对rhIL-877的加工过程总结：展示了经转化产物诱导的中性粒细胞脱颗粒后，回收的总IL-8（方块）、IL-877（圆圈）及MMP-9（三角）的浓度。横轴为孵育时间（小时），纵轴为IL-8/MMP-9相对于0小时样本的倍数变化。数据点为3次独立实验的平均值（±SEM）。各时间点的浓度与缓冲液对照组的差异采用双因素方差分析结合邦费罗尼事后检验进行比较（*** = p<0.001）。补充图S10：纯化蛋白酶3对rhIL-872的加工过程。在24小时孵育期间，分别与缓冲液（圆圈）及蛋白酶3（方块）共孵育后，总IL-8的回收率（以0小时浓度的倍数变化表示）。横轴为孵育时间（小时），纵轴为浓度倍数变化（相对于“0小时”样本的浓度）。数据点为3次独立实验的平均值（±SEM）。浓度差异采用双因素方差分析结合邦费罗尼事后检验进行比较（*** = p<0.001）。(DOC)