A prospective investigation of interleukin-8 levels in pediatric acute respiratory failure and acute respiratory distress syndrome

The statistical analysis was performed using SAS version 9.4. Development of PARDS, death, duration of mechanical ventilation, and length of PICU stay in survivors were the outcomes examined for association with plasma IL-8 levels across all days and if significant with IL-8 levels on each individual day. Due to the skewed distribution of plasma IL-8 levels, a natural log transformation was used for the analyses. Basic univariate (including frequency distributions or means, medians and standard deviations, analysis of variance, Wilcoxon rank-sum tests) and bivariate (including chi-square, Fisher’s exact, t tests, Pearson correlations, or longitudinal models, as appropriate) analyses of all demographic measures, clinical measures, and outcomes were conducted across all study days and on each individual study day (0, 1, 2, and 3). Comparisons with respect to these variables used generalized estimating equations (GEE) to account for correlations within individuals. Study hypotheses were evaluated using longitudinal models across days and if significant, with separate models for each day using logistic regression models for binary outcomes, Poisson regression models for the duration of mechanical ventilation, and linear regression for the length of PICU stay. The results of the bivariate analyses and forward stepwise linear models were used to develop the final multivariate models. The potential covariates of age, gender, race, ethnicity, prior medical co-morbidity (including the history of prematurity, asthma, seizure disorder, neurological disorder, cancer, or immune deficiency), primary diagnosis associated with acute respiratory failure, and PRISM III score were considered for the multivariate models. For the final models, each outcome was modeled as a function of age, PRISM III score, diagnosis of sepsis, and IL-8 level, with study day included when longitudinal models across all days were evaluated. The diagnosis of sepsis was correlated to a past medical history of cancer and of immune deficiency; therefore, only sepsis was included in the final model. All multivariable logistic regression models of the binary outcomes accounted for individual clustering using GEE and adjusted for the covariates to assess the association of IL-8 across all days with outcomes. Final models for each individual day included IL-8 as a fixed covariate, whereas when examining IL-8 across all days, IL-8 was included as a time-varying covariate. The GEE analysis across all days included only patients with two (n = 143) or three (n = 208) IL-8 measurements within the time frame of interest; 73 patients with only one measurement were not included in the analysis. The total number of patients included in analyses done on each day is indicated in the figures, or is shown in table legends, and includes the 73 patients that were dropped from the analyses done across all days. We reported odds ratios with 95% confidence intervals. All statistical tests were two-tailed, with a significance level of 0.05.

DNA was extracted from whole blood using the Wizard Genomic DNA Purification Kit (Promega, Madison, WI). Ninety-two percent of the samples (n = 481) had sufficient DNA for genotyping which was done using a custom Illumina Golden gate panel which included the three IL-8 SNPs of interest, rs4073, rs2227306, and rs2227307. SNP call rates were 100%, and genotypes did not deviate from Hardy-Weinberg equilibrium. Genotyping was done blinded to the clinical status of individuals. The association of these genetic variants with PARDS was examined as described previously [20]. Briefly, the cohort was stratified by race and ethnicity into three major subgroups, non-Hispanic Caucasians, Hispanic Caucasians, and African Americans (52%, 20%, and 17% of the cohort, respectively). Association of IL-8 SNPs with PARDS within each group was examined, and then, results of the individual subgroups were combined and examined by meta-analysis using the program METAL [22].

Of the 549 patients enrolled in BALI, 69% (n = 378) developed PARDS. Within the PARDS cohort, 83% met the diagnosis for PARDS at the time of intubation (day 0) and 94% by day 1. Plasma IL-8 levels were higher in children with PARDS compared to those children without PARDS on all days measured, but when examining each day, achieved statistical significance only on day 2 (p = 0.02 on day 2, Fig. 1a). Multivariable analyses adjusted for age, PRISM III, and sepsis diagnosis demonstrated that plasma IL-8 was not independently associated with PARDS across all days or on any specific day (Additional file 1: Table S3).

On bivariate analysis of this cohort of children with acute respiratory failure, plasma IL-8 was significantly higher in non-survivors on each day measured (Fig. 1b, p < 0.05) with median IL-8 levels 4–12 fold higher in non-survivors than survivors. As IL-8 has been reported to be associated with death in children with PARDS, the cohort was stratified by PARDS to examine whether PARDS patients were responsible for the higher IL-8 levels observed in non-survivors. As shown in Fig. 2a, the levels of IL-8 were significantly higher in survivors with PARDS compared to survivors without PARDS; however, the levels of IL-8 were not significantly different in non-survivors with and without PARDS (Fig. 2b) but are higher than those seen in survivors with or without PARDS (Fig. 2b vs a) suggesting that IL-8 is associated with death in patients with acute respiratory failure independent of whether the child has PARDS. On multivariate analysis with adjustment for severity of illness (PRISM III), age, and sepsis diagnosis, plasma IL-8 was independently associated with death on days 1, 2, and 3 (Table 2). When PARDS was added to the model, IL-8 was still independently associated with death (Additional file 1: Table S4) again suggesting that IL-8 is associated with death both in the whole cohort and in patients without PARDS.

Bivariate analysis also indicated that plasma IL-8 was significantly correlated with duration of mechanical ventilation on each day measured and with PICU length of stay on days 1, 2, and 3 (Additional file 1: Table S5). When duration of mechanical ventilation was dichotomized into 7 or more ventilator days versus fewer than 7 days (median duration of mechanical ventilation was 7.1 days, IQR, 4.0–13.6), plasma IL-8 was significantly higher in patients with a duration of mechanical ventilation ≥ 7 days on each day measured (p < 0.01, Fig. 3a). Using 14 days to dichotomize PICU length of stay (median PICU length of stay was 10.6 days, IQR, 6.6–18.4), IL-8 was significantly elevated on each day measured in those patients requiring a prolonged PICU stay (14 or more days vs fewer than 14 days, p < 0.01 on each day, Fig. 3b). Using Poisson regression and controlling for age, severity of illness, and sepsis diagnosis, plasma IL-8 was independently associated with prolonged duration of mechanical ventilation on each day measured (p ≤ 0.01 on each day). Finally, plasma IL-8 was associated with prolonged PICU length of stay in survivors on each day measured when adjusted for age and severity of illness and was also associated with longer length of stay on days 0 (p = 0.01), 2 (p = 0.02), and 3 (p = 0.03) when sepsis diagnosis was also added to the model (data not shown).

Three IL-8 SNPs previously reported to be associated with severity in cystic fibrosis (rs4073, rs2227306, rs2227307) or with the length of mechanical ventilation in adults with ARDS (rs4073) were examined for association with PARDS. The cohort was stratified into non-Hispanic Caucasians, Hispanic Caucasians, and African Americans for analyses which were done with adjustment for population structure (as described under the “Methods” section). None of the variants examined showed a significant association with PARDS within any of the subgroups or when the three subgroups were combined in a meta-analysis (n = 412). In addition, these variants were not associated with plasma levels of IL-8.