Traumatic injury with its potential for infection was likely a common cause of death for our human ancestors. Even today, massive injury remains the most common cause of death for those under the age of 45 yr in developed countries (Sasser et al., 2006; Probst et al., 2009). Only recently has the human injury response been studied systematically at the genomic level and only now is it beginning to become better understood. Unfortunately, billions of dollars worldwide have been invested on new biological therapeutics for severe injury, as well as for its sequelae, sepsis and septic shock, with disappointing, if not harmful, results. The current immune, inflammatory paradigm, based on an incomplete understanding of the functional integration of the complex host response, remains a major impediment to the development of effective innovative therapies. Prior work has focused on the role of individual mediators (e.g., TNF or IL-1; Giannoudis, 2003; DeLong and Born, 2004; Giannoudis et al., 2004; Keel and Trentz, 2005) or processes such as apoptosis and cellular death in nosocomial infections and organ injury after trauma (Hotchkiss et al., 2009). Rather than using a reductionist approach, we examined the genome-wide expression patterns of blood leukocytes in the immediate postinjury period to better understand the overall priorities and patterns of gene expression underlying not only the initial injury response, but also the development of complications and delayed clinical recovery (Flohé et al., 2008). We have compared the genomic response by blood leukocytes to trauma with the changes in gene expression produced by major burns (>20% of body surface area), as well as the response by healthy subjects to the administration of low-dose bacterial endotoxin (Calvano et al., 2005). The results of this systems-wide approach to the study of severe human injury challenge several current clinical dogmas regarding the nature of the host response to severe injury. In addition, the datasets described in this report of our large clinical study are an important resource that will enable important future analyses like mathematical modeling and predicting patient outcomes. Circulating blood leukocytes have the capacity to seek out, recognize, and mount an appropriate inflammatory response at the earliest sign of injury. Innate immune cells initially recognize and are activated by pathogen-associated molecular patterns (PAMPs) or endogenous alarmins and danger signals (Xu et al., 2009; Puneet et al., 2010; Zhang et al., 2010). Blood neutrophils, monocytes, and NK cells are implicated as primary effectors during the initial inflammation and activation of innate immunity. Severe trauma has also been characterized by immunosuppression, primarily seen on the adaptive immune system with T lymphocyte populations being the most markedly affected cell population (Hotchkiss and Karl, 2003; Keel and Trentz, 2005). Although antiinflammatory processes and reduced effector T cell function are necessary to limit or localize the response to severe trauma, a prolonged or exaggerated period of immune suppression or defective immune response leads to increased susceptibility to secondary infections (Hotchkiss and Karl, 2003). We isolated whole blood leukocytes and performed genome-wide expression analysis from the Affymetrix U133 GeneChip using a cohort of 167 patients between the ages of 18 and 55 yr who consented to blood sampling from 1,637 adult severe blunt trauma patients who developed hypotension or acidosis and required resuscitation with blood products from seven US hospitals. Blood was sampled within 12 h and at 1, 4, 7, 14, 21, and 28 d after the injury. Genome-wide expression from patients with trauma was compared with age-, sex-, and ethnicity-matched healthy subjects and with 133 adult patients after severe burn injury (>20% of the body surface area) or 4 healthy adult subjects administered low-dose bacterial endotoxin. There were multiple objectives of the present study. The first objective was to determine whether the multiple-organ dysfunction syndrome (MODS) phenotypes observed agreed with the current paradigm that explains whether the MODS seen after injury is the result of excessive proinflammatory responses (systemic inflammatory response syndrome [SIRS]) followed temporally by compensatory antiinflammatory response syndrome (CARS) and suppression of adaptive immunity. In those with MODS, this period of recovery from organ failure varied from a few days, nonrecovery at 28 d, or death. Unexpectedly, there were no clinical outcomes consistent with MODS followed by recovery and subsequently severe MODS that might be predicted as a second hit (Sauaia et al., 1994; Keel and Trentz, 2005). The second objective was to determine whether there were recognizable gene expression changes in the blood leukocytes after severe blunt trauma. Our data indicate that severe trauma altered the expression of >80% of the leukocyte transcriptome during the first 28 d after injury, and these changes were highly reproducible within at least 30 discernable gene expression patterns. Of the most significantly regulated pathways, injury produced early activation of those involving innate and simultaneous suppression of those involving adaptive immunity. Interestingly, severity of injury, magnitude of physiological derangement, and volume of transfused blood minimally affected these patterns. The third objective was to determine whether there were patterns of gene expression associated with two extremes of clinical recovery (uncomplicated versus complicated). Surprisingly, gene expression patterns were highly comparable between these two recovery extremes with selective differences in only magnitude and duration. Our data support a new paradigm for the host immunological response to injury. RESULTS AND DISCUSSION Trauma patients versus healthy subjects The characteristics of the trauma patients and healthy subjects and the patient clinical outcomes are shown in Table I and Fig. 1 A. As seen in Fig. 1 A, the majority of trauma patients presented with mild to severe MODS but recovered before 28 d. There was only a small fraction of patients who either did not develop MODS or developed severe MODS and did not recover before 28 d. There were no patients who developed MODS initially, partially recovered, but went on to develop severe MODS. Table I. Characteristics and outcomes in the 167 trauma patients and 37 healthy control subjects Parameter Controls (n = 37) Total cohort (n = 167) Uncomplicated recovery patient ( 80% of the human genome over the first 28 d (using a false discovery rate [FDR] adjusted probability 14 d, no recovery, or death; Fig. 1 A). We were interested to identify whether the genomic patterns were different between patients with complicated and uncomplicated outcomes. That is, are there genes or pathways that behave differently in these extremes in clinical recovery? There were 2,391 genes in the circulating leukocytes whose expression was significantly different (FDR 20% of the total body surface area and from 4 healthy humans after administration of low-dose bacterial endotoxin (Calvano et al., 2005). Additional information. A supplemental web-based portal (Massachusetts General Hospital, 2011) is available to explore in greater detail the largest clinical and genomic database to date from severely injured humans. Data in this study have been deposited in the GEO DataSets site under accession number GSE11375. Online supplemental material. Fig. S1 shows the distribution of 167 samples assayed within the first 12 h of injury. Fig. S2 shows the comparison of gene expression patterns of the 5,136 genes with a greater than twofold change from control subjects for patients with a complicated (n = 55) and uncomplicated (n = 41) clinical recovery. Fig. S3 presents data on the plasma cytokine/chemokine levels after severe blunt injury. Fig. S4 shows the effect of transfusion, ISS, and base deficit on early patterns of gene expression. Fig. S5 presents data on the concentrations of blood leukocyte populations in the trauma patients. Fig. S6 depicts the differences in gene expression patterns between patients with a complicated and uncomplicated recovery. Online supplemental material is available at http://www.jem.org/cgi/content/full/jem.20111354/DC1.
