Search

Major trauma currently accounts for 10% of deaths globally. Although the implementation of standardised protocols for the management of poly-trauma has resulted in significant improvements in early-phase trauma care, sepsis and multiple organ dysfunction syndrome remain challenging complications contributing to significant mortality. The immune response to trauma is postulated to follow a 'two-hit' course, with clinical outcomes being dictated through a complex interplay between cascades of systemic pro-inflammatory and compensatory anti-inflammatory responses (SIRS and CARS). The desired outcome, homoeostasis, is the resultant balance between the two opposing arms of the immune response. It is postulated that tissue damage due to over-activity of the SIRS response and immune-paresis due to over-activity of the CARS response render patients susceptible to nosocomial infections (sepsis) with multiple organ dysfunction (MODS)(MOD) and poor clinical outcomes (increased mortality and morbidity). The ability to detect early markers of immune imbalance would enable the identification and stratification of the cohort of patients likely to have poor clinical outcomes for targeted and individualised clinical interventions. The objective of the study is to test the hypothesis that immune markers related to an overactive counter-regulatory immune response could predict immuno-paresis and susceptibility to nosocomial infections contributing to poor clinical outcomes (increased length of stay/sepsis/MODS/mortality). Of particular interest, is the role of a subset of CD4+ regulatory T lymphocytes expressing CD25+, CD25Bright, CD127Low and FoxP3+ immune markers, as potential biomarkers for poor clinical outcome following major trauma. We present here data from a prospective observational study of major trauma patients (n=80) recruited to the Central Manchester Foundation Trust (CMFT) and Salford Royal Foundation Trust (SRFT). Serial blood samples (Day 1, 3 and 5 post-trauma) were processed for the expression of T lymphocyte surface markers (CD4+, CD3+, CD8+, CD25+, CD127Low, and the intracellular transcription factor (FoxP3)) in relation to clinical and biochemical data collected in parallel. Preliminary processes were aimed at optimising and validating an experimental methodology to detect markers of the suppressive immune response, namely the CD25Bright, CD127Low, and FoxP3+ lineages. A significant high positive correlation was seen in the trends between CD25Bright and CD127Low with R values 0.975, P<0.01 confirming alternate routes xxiii to capture the immunosuppressive Treg subset of interest. The correlation between FoxP3+ and the two surface markers (CD25Bright and CD127Low) were less marked, confirming the mixed population of Treg cells likely to be captured in the CD4+FoxP3+ lineage. In defining trauma-induced trends (Day1, 3, 5 post-trauma) in T cell subset populations, a significant increase was observed in CD4+ /CD8+ ratios (P=0.013) alongside a reducing trend for CD8+ T cell subsets (P=0.008). The nTreg (CD25Bright, CD127Low, FoxP3+) trends showed a decrease over Days 1 to 5 post-trauma. While this was not statistically significant, the averaged values for all days were higher than averages described for normal populations. A range of clinical parameters was defined as indicators of poor clinical outcome, including, Sequential Organ Failure Scores (SOFA), Antibiotic usage, Serum lactate and C-reactive protein (CRP) levels and mortality. Tcell subsets were compared with the poor outcome parameters in a bid to define trauma-induced trends and potential predictions for poor outcome. Average values for CD3+, CD4+ subsets and CD4+/CD8+ ratios were lower in patients with higher SOFA scores and a reverse correlation was observed with CD8+ T lymphocyte subsets (P > 0.05). When averaged nTreg subset values were compared against SOFA clustering, SOFA scores ≥ 6 on admission were associated with a statistically significant increase (CD4+CD25Bright and CD4+CD25+CD127Low subsets; P=0.002 and P=0.004, respectively). When data were analysed to map positive predictive correlations between early nTreg changes and late (Day 5/8) poor clinical outcome parameters (SOFA score), a weak positive but the statistically significant correlation was observed (CD4+CD25Bright with SOFA score Day 5 and 8, (R=0.259, P=0.037 and R=0.284, P=0.022, respectively). Antibiotic usage failed to correlate with SOFA scores on Days 5 and 8 emphasising a need for definitive markers to guide antibiotic usage policies. A weak positive correlation was observed between CD8+ T cells and CRP at Day 5, R=0.588, P<0.01 and a weak negative correlation between CD4+/CD8+ ratio and CD4+ T cells and CRP at Day 5, (R=-0.459, P<0.01, and R=-0.557, P<0.01, respectively). A significant weak positive correlation was observed in trauma patients T cells (CD3+CD4+, and CD4+/CD8+ ratio) with lactate Day 5, (R=0.394, P<0.05 and R=0.425, P<0.05, respectively) and a significant weak positive correlation between CD4+FoxP3+ Treg cells and lactate levels at Day 5, R=0.474, P<0.01. The mortality rate in the study group was 8.75% and correlated to rates in published literature. xxiv The results showed that high nTreg cell subsets and CD8+ T cells could be the potential markers of poor clinical outcome in major trauma patients. The data reported in this study will be validated in the ongoing Phase II trauma biomarker study for statistical powering in a study cohort of 200 patients.