Major traumatic injury : serum cytokine biomarkers as predictors of poor clinical outcome

Ramaswamy, DS

Abstract

Trauma is a leading cause of mortality and morbidity worldwide, accounting for about 5.8 million deaths annually. England alone sees approximately 25,000 cases of major trauma each year and over a fourth of these result in loss of life.
Death due to trauma is time factorial and bimodally distributed. The first phase appears within an hour of injury, presenting immediate traumatic effects due to fatal injuries and haemorrhagic shocks. The second phase is marked by onset of complications after a week, characterized by further complications such as sepsis, multiple organ dysfunction and multiple organ failure. This is caused by an imbalance between pro-inflammatory and anti-inflammatory responses to traumatic injury, largely orchestrated by cytokines.
Cytokines are known, to be predominantly secreted by helper T cells and activated monocytes and macrophages and numerous distinct cell types synchronize their role as part of the immune system. Each of these cell types has a distinct role in the immune system and communicates with other immune cells using secreted cytokines. Interleukins are a large class of cytokines, which are involved in systemic inflammation and immune system modulation by stimulating humoral or cell-mediated immune responses. They play an important role in fighting infection and diseases. Interleukins are also predictive biomarkers whose concentrations are measured through multiplex bioassays.
The research study aims at investigating a panel of serum cytokines, cellular and metabolomic markers as a potential predictor of poor clinical outcome in major trauma. The study is a continuation of a research project with a larger cohort of 200 patients selectively recruited from Central Manchester Foundation Trust (CMFT) and Salford Royal Foundation Trust (SRFT).
The aim of the study was to investigate the potential for a panel of cytokines to predict clinical outcome in patients with major traumatic injuries. This was achieved by measuring the concentrations of interleukin-13 and interleukin-17 through cytometric bead array methods on which statistical analysis was performed. The study analysis of the sub-cohort of 30 patients, investigated the role of IL-13 and IL-17 incepting single organ failure or MOF and evaluating them as candidate biomarkers for clinical prediction of good and poor outcomes.
In this study, IL-13 concentration increased between day 1 and day 5, post trauma (p=0.02). Based on the Sequential organ failure assessment scores (SOFA), day 1 concentrations were compared to day 5 and day 8 at 2 different cut-offs, namely SOFA score <3 and ≥ 3 and SOFA score <6 and ≥6. This stratification showed no statistically significant differences meaning SOFA cut offs did not account for a clinical outcome prediction. Later Delta SOFA (day 5 SOFA – day 1 SOFA) was employed to assess whether the patients improved or got worse at the ICU. Though the comparison between IL-13 concentration on day 1 and delta SOFA did not show statistical significance, it revealed a weak negative relationship between them (r=-0.318, p=0.087), indicating that the patients got better at the ICU. Although IL-13 data did not show statistical significance based on SOFA, the data revealed a good clinical outcome when it was clustered tightly based on the movement of SOFA across concentrations.
A percentage concentration analysis was conducted between day 1 and day 5 concentrations to show how IL-13 levels changed at different SOFA scores, and whether they could predict clinical outcome. The increase in average IL-13 concentration on D5 by 10% (3 pg/ml), tallied with decreased SOFA score, thereby indicating good clinical outcome. The analysis of IL-17 concentration showed a marginal decrease between day I and day 5 without a significant statistical difference (p=0.994). The concentration of IL-17 in day 1 was tested against SOFA score calculated for day 5 and day 8 at two different cut-offs, namely SOFA score <3 and ≥ 3 and SOFA score <6 and ≥6.
At a threshold value of 3, day 1 IL - 17 concentration, statistically correlated with SOFA score in day 5 at p=0.048. The IL-17 concentration against SOFA score in day 8 returned similar results at the cut off 3 with p value 0.042 with a statistical significance. This shows that IL-17 levels on the day of admission could predict the onset of single organ failure on case day 5. Repeating the clustering for day 1, IL-17 concentration with day 8 SOFA score at threshold 6 showed statistical significance of p=0.044, indicating the onset of multiple organ failure (MOF).
The correlational analysis between IL-13 day 1 and IL-17 day 1 concentrations and CRP day 5 showed no association. In this study, the cytokine levels were defined using CRP day 5 levels instead of SOFA scores, the data was not statistically significant. This could be because of the small sample size (n =16) made available. The CRP levels ranged between 1.1 mg/L to 197 mg/L for these 16 patients. Similarly, the average lactate concentration varied from
3.863 mM/L on day 1 to 1.010 mM/L on day 5 for only 8 data points. This extreme variation could have skewed the sample and resulted in not obtaining statistical significance.
A cross sectional comparison was conducted amongst the multiplex panel of cytokines involving on a common patient cohort (N=30) derived from the pilot project cohort (N=200). IL-13 and IL-17 on day 1 and day 5 were correlated with IL-4, IL-8, and IL-12.
The cluster plots between IL-13 and IL-17 on day 1 and day 5 showed a strong positive linear co-relationship (r = 0.575 & 0.450) respectively, expressing strong positive feedback loop between IL-13 and IL-17. The correlation analysis between IL-13 and IL-4 levels on day 1 revealed a strong positive linear association between them at r=0.537. The correlational analysis between IL-13 and IL-8 revealed negative correlations on day 1 and day 5 (r=--0.030 and r=-0.353) respectively. IL-13 and IL-12 showed a weak positive correlations on day 1 and day 5 at r=0.376 and 0.321, respectively.
The concentrations of IL-17 and IL-4 and day 5 showed a weak positive linear relationship at r=0.356 on day 1 and a strong positive correlation at r=0.518 on day 5. Correlation between IL-17 and IL-8, revealed a weak positive relationship (r=0.032) and on day 5 steeped to -0.226, indicating negative linear relationship. IL-17 and IL-12 concentrations on day 1, strongly correlated at r value 0.552. The analysis of expression levels of this spectrum of 5 cytokines indicates their synergistic relationships, interwoven in positive or negative loop of feedback mechanisms.
The results of this study show that interleukin concentrations could provide an early prediction of complications and could offer a promising direction for effective therapeutic breakthroughs.

Citation

Ramaswamy, D. Major traumatic injury : serum cytokine biomarkers as predictors of poor clinical outcome. (Dissertation). University of Salford

• Thesis Type: Dissertation
• Deposit Date: Dec 20, 2021
• Publicly Available Date: Dec 20, 2021
• Award Date: Apr 23, 2021