Defining and predicting the innate immune response to critical injury

Trauma is a leading cause of death worldwide with over 5 million deaths/year, and is responsible for 14% of the global burden of disease. Although patients may survive their initial injury, the massive associated tissue damage often results in later immune system-related clinical complications such as recurrent prolonged infection and multiple organ dysfunction syndrome (MODS), which is failure of organ systems such as lung, kidney, liver and heart. In England there are over 3,300 trauma patients with MODS every year, who must be managed on intensive care units, occupying 33,000 critical care bed days, and despite which, 25% will die. Those who survive are often left with long-term consequences of these complications. Currently the processes which cause some patients to develop severe clinical complications are largely unknown.

Normally the immune system is activated to clear damaged tissue and prevent infection from taking hold. However, modern trauma care now resuscitates patients with massive physical injuries, which humans could never have evolved to respond to appropriately. The immune response is no longer limited to the sites of injury, but instead becomes disordered and itself damages distant organs like the lungs, heart, kidneys and liver. Although these problems do not become clinically apparent for 1-2 days after trauma, we have shown that immune responses are set within the first two hours after injury (the hyper-acute window).

The hyper-acute period is a critical time in a trauma patient's care, and conducting parallel research is extremely challenging in this environment. For this reason, very little is known about the immediate response of the human immune system to trauma. Our Centre has focused our research into this critical time-point and we have embedded a specialist research team within our Major Trauma Centre, who work alongside the clinical teams.

The central question in contemporary trauma immunology, is whether the immune response to injury that leads to clinical complications is simply a magnified response to massive tissue damage above a critical threshold, or whether some patients have an abnormal response which results in adverse outcomes. Our proposed research is designed to answer this key question.

Our first aim is to understand the relationship between tissue damage and activation of the immune system. We will characterize the transcriptome (a read-out of genes that are switched on) of circulating white blood cells in a large cohort of patients with different levels of injury. Importantly, we will assay objective indicators of cell damage (rather than use a subjective clinical assessment) to understand the correlation between magnitude of tissue damage, immune cell activation, and the development of adverse outcomes. With this as a baseline, we will use modern data analysis techniques to understand how variation in different injury sub-groups lead to divergent clinical trajectories.

Our second aim is to specify in detail these aberrant responses to severe injury. We hypothesize that poor outcomes result from specific changes in how cell damage is perceived by the immune system; and from changes in signaling responses in a specific immune pathway mediated through IL-6/JAK2/STAT3. Both mechanisms were identified as potentially important from our original pilot study of the hyper-acute response to injury.

We believe findings in this study will prove central to further developments in the field. The unique data and knowledge generated by this project will provide a new insight into this critical time period for trauma patients. These will lead to new prediction tools, diagnostics and management strategies. Ultimately the identified mechanisms will form the basis for translational research and novel therapeutics that could be delivered in the hyper-acute phase, at the road-side or in A&E, to protect patients from later complications and improve survival.

Samples: This research will use existing and future blood samples from critically injured patients (ISS 25+) collected at the Royal London Major Trauma Centre in the "hyper-acute window" (within 2 hours of injury). Samples are processed for plasma, protease-inhibited plasma, and leucocytes (as PAXGENE and buffy coat). 676 existing samples meeting study criteria are available.

Methodologies: Aim 1: To understand the relationship between tissue damage and advserse clinical outcomes by;
1.1: Whole genome transcriptomic analysis using RNA sequencing on samples taken in the hyper-acute window
1.2: Measurement of objective intrinsic indicators of tissue damage and inflammation-associated cytokines/chemokines
1.3: Latent class mixed modelling machine learning approaches combined with support vector algorithms

Methodologies: Aim 2: To gain mechanistic insight into differential hyper-acute immune responses to trauma, by;
2.1: Single cell immune cell profiling using mass spectrometry (cyTOF) on a prospective cohort
2.2: Measurement of plasma levels of IL-6, sIL-6R, and sgp130 in our new cohort
2.3: 10X Genomics single-cell-RNA-seq platform to generate single-cell transcriptomic data for 5-10K purified PBMCs to identify differentially regulated biomarkers and critical signaling modalities
2.4: Flow cytometry & functional analyses of leukocytes from healthy volunteers cultured with hyper-acute plasma from trauma patients who subsequently developed (or did not) MODS

Exploitation of results: Key applications will be; A) The generation of new knowledge in understanding the human immune response to tissue damage and its relationship with MODS and immunosuppression; B) Hyper-acute biomarkers and stratification tools for prognostication, clinical trials and precision approaches to resuscitation; and C) novel targets for therapeutic repositioning and drug discovery to reduce the incidence and severity of organ dysfunction and infection in trauma patients.

Nationally in the UK there are over 3,300 trauma patients on critical care with multiple organ dysfunction syndrome (MODS) and immunosuppression every year, requiring 33,000 critical care bed days, of which 25% will still die.

Ultimately, we expect the proposed research to be primarily of benefit to trauma patients, to reduce the enormous mortality and morbidity associated with a dysregulated immune response to critical injury. If the research can reduce the incidence and severity of MODS this will dramatically reduce healthcare costs and resource utilization for these patients. Furthermore, if infections can be reduced then antibiotic utilization and drug resistance may also be decreased. This will be especially important in low and middle-income countries where access to critical care is limited. We believe benefits can translate to improved patient outcomes within 5 to 10 years of the start of the project. Improved quality of life for patients and families and return to work will provide broad benefits for the NHS, global health and society.

There will be important opportunities for the commercial sector derived from this knowledge. Our preliminary data has already led us to a Phase IIa repositioning trial of Artesunate to reduce MODS in trauma, putatively by augmenting the action of cell survival pathways. Therapeutic opportunities may also be identified for the prevention or treatment of post-traumatic immunosuppression. The data produced will be ideal for repositioning programmes or the development of novel therapeutics using chemogenomic and similar methodologies. There will likely also be a range of new diagnostic opportunities with the identification of biomarkers in the hyper-acute window as prognostic tools and for stratified medicine approaches to individualise care.

We believe the programme will also provide a strong model for clinical trauma research and research into critical illness in general. The provision of a dedicated, embedded research facility and team within a hospital is becoming less common, but we have already demonstrated the power of this approach with our work on coagulopathy. The proposed project will lead to a step-change in the embedded laboratory's research capability and will demonstrate the ability of such units to achieve impactful change in short timeframes. By disseminating our research team's processes and practices to other trauma centres, nationally and internationally, we will be able to spread not only research findings but also good research practice and methodologies for the emergency research environment.

Human biology education and science engagement programmes can also benefit from this programme. Trauma is hugely engaging and we have a successful programme with schools, in part through our Centre of the Cell public engagement program, to use trauma research (currently in bleeding and coagulopathy) to engage students in science, to stimulate career ideas, and for education around human biology. We have exhibited at the Big Bang Fair and the Royal Society's Summer Science exhibition. We have invested in a new exhibit "So you think you're Immune to Trauma?" and will promote and develop this over the course of the project. We believe an in-depth understanding of leukocyte responses to injury could also serve as a platform for education and engagement in ideas around biology, evolution, big data and complex systems.