Rapid systemic surge of IL-33 after severe human trauma: a prospective observational study

Background Alarmins are considered proximal mediators of the immune response after tissue injury. Understanding their biology could pave the way for development of new therapeutic targets and biomarkers in human disease, including multiple trauma. In this study we explored high-resolution concentration kinetics of the alarmin interleukin-33 (IL-33) early after human trauma. Methods Plasma samples were serially collected from 136 trauma patients immediately after hospital admission, 2, 4, 6, and 8 h thereafter, and every morning in the ICU. Levels of IL-33 and its decoy receptor sST2 were measured by immunoassays. Results We observed a rapid and transient surge of IL-33 in a subset of critically injured patients. These patients had more widespread tissue injuries and a greater degree of early coagulopathy. IL-33 half-life (t1/2) was 1.4 h (95% CI 1.2–1.6). sST2 displayed a distinctly different pattern with low initial levels but massive increase at later time points. Conclusions We describe for the first time early high-resolution IL-33 concentration kinetics in individual patients after trauma and correlate systemic IL-33 release to clinical data. These findings provide insight into a potentially important axis of danger signaling in humans. Supplementary Information The online version contains supplementary material available at 10.1186/s10020-021-00288-1.

modification of the injury severity score (ISS), an overall anatomical injury scoring system according to which each injury is assigned an abbreviated injury scale (AIS) score and allocated to one of six body regions. The 3 most severely injured body regions have their most severe AIS score squared and added together to produce the ISS. By contrast, NISS is defined as the sum of squares of the AIS scores of each of a patient's three most severe injuries regardless of body region. NISS is a better predictor of post-injury multiple organ failure and mortality than ISS but a less accurate measure of total tissue injury volume(3).
Base excess (BE) is a useful surrogate marker of hypoperfusion, and several studies have shown an exponential increase in mortality in patients with an admission BE< -6. In our cohort measurement of BE was missing in 9 patients.

IL-33 Bio-plex analyses
Analyses for IL-33 in 1094 patient plasma samples and 20 control samples were performed with a bead-based immunoassay using the Bio-Plex Pro Human Th17 Cytokine IL-33 set (Bio-Rad Laboratories, Hercules, CA) and analyzed with a Bio-Plex MAGPIX multiplex reader according to the manufacturer's instructions. All plasma samples were diluted 1:4 before analysis. Lower detection limit (LDL) in this immunoassay ranged from 1.7-4.4 pg/ml within different runs, and samples that were below LDL were assigned 0 pg/ml.

IL-33 Immunoprecipitation
4 µg of an anti-IL-33 rabbit antibody (raised against the first 15 amino acids of the Nterminal) were bound to 50 µl sheep anti-rabbit Dynabeads (Life Technologies, Oslo, Norway) by end-to-end rotation for 40 min at 4 °C. 400 µl serum from trauma patients (diluted 1:2 in PBS) or 20 µg human umbilical vein endothelial cells (HUVEC) lysate (diluted in 800 µl PBS) were incubated with the antibody-beads for 16 h at 4 °C.
Membrane was washed as previously described before addition of ECL western blotting substrate (Life Technologies, Oslo, Norway)

ST2 ELISA
Analyses for ST2 were performed using the Human ST2/IL-1 R4 Quantikine ELISA kit (R&D Biosystems, Minneapolis, MN). The antibodies used in this ELISA detects both membrane-bound ST2 (ST2L) and soluble ST2 (sST2), but in cell-free plasma/serum samples it is considered an accurate measure of sST2. A selection of samples from the cohort was chosen (18 patients and 6 controls) in order to include survivors/non-survivors, IL-33neg/IL-33pos and patients with different injury severity. Due to large increases in sST2 levels after trauma that extended beyond the dynamic range of detection of the ELISA, all samples were run in duplicates in two different dilutions (1:40 and 1:1000). Concentration values from samples with low levels of ST2 (<50 ng/ml) were obtained from the low dilution, and similarly values from samples with high levels (>50 ng/ml) were obtained from the highly diluted replicates. A perfect dilution linearity performance of the ELISA as described by the producer was not observed in our hands. This incongruence therefore represents some inaccuracy in absolute concentrations when using values obtained from two different dilutions but it had no impact on the overall shape of the time curves shown in Figure 3.

Routine laboratory tests
Standard laboratory tests, including aPTT, INR and base excess, were performed routinely at admission, and at later times points either as part of routine measurements in the ICU or at clinicians' discretion. Neither CK nor LDH were routinely obtained on admission, but maximum levels within first 48 hours after injury are shown in table 1. All analyses were performed according to standard operating procedures at the department of medical biochemistry, Oslo University Hospital. As part of this research project we have access to electronic records of all the laboratory analyses. 2. Osler T, Baker SP, Long W. A modification of the injury severity score that both improves accuracy and simplifies scoring. J Trauma 1997;43(6):922-5-discussion 925-6.

P a t i e n t 1 T 1 P a t i e n t 3 T 1 P a t i e n t 1 T 2 P a t i e n t 3 T 2 P a t i e n t 2 T 1 I P H U V E C l y s a t e P a t i e n t 2 T 2 H U V E C l y s a t e ( p o s c t r l )
Supplemental Figure 1 Supplemental figure 1 Immunoprecipitation of samples from 3 patients with detectable IL-33 at admission as determined by bead-based immunoassay. Two samples from each patient were analyzed; an admission sample (T1) with high IL-33 level and one subsequent sample where the IL-33 level as determined by immunoassay was low or undetectable (T2). Immunoprecipitation of lysates of human umbilical vein endothelial cells (HUVECs) and direct detection of IL-33 in HUVEC lysate were included as controls.