Severe polytraumatic injury initiates a robust immune response. Broad immune dysfunction in patients with such injuries has been well-documented; however, early biomarkers of immune dysfunction post-injury, which are critical for comprehensive intervention and can predict the clinical course of patients, have not been reported. Current circulating markers such as IL-6 and IL-10 are broad, non-specific, and lag behind the clinical course of patients. General blockade of the inflammatory response is detrimental to patients, as a certain degree of regulated inflammation is critical and necessary following trauma. Exosomes, small membrane-bound extracellular vesicles, found in a variety of biofluids, carry within them a complex functional cargo, comprised of coding and non-coding RNAs, proteins, and metabolites. Composition of circulating exosomal cargo is modulated by changes in the intra- and extracellular microenvironment, thereby serving as a homeostasis sensor. With its extensively documented involvement in immune regulation in multiple pathologies, study of exosomal cargo in polytrauma patients can provide critical insights on trauma-specific, temporal immune dysregulation, with tremendous potential to serve as unique biomarkers and therapeutic targets for timely and precise intervention.

Background: Assessment of immune status in critically ill patients is often based on serial tracking of systemic cytokine levels and clinical laboratory values. Exosomes are extracellular vesicles that can be secreted and internalized by cells to transport important cellular cargo in the regulation of numerous physiological and pathological processes. Here, we characterize the early compartmentalization profile of key proinflammatory mediators in serum exosomes in the steady state and following trauma. Adult male Sprague-Dawley rats (91 including naïve) were divided into one of four traumatic injury model groups incorporating whole-body blast, fracture, soft-tissue crush injury, tourniquet-induced ischemia, and limb amputation. Serum was collected at 1, 3, 6, and 24 h, and 3- and 7-day post-injury. Electrochemiluminescence-based immunoassays for 9 key proinflammatory mediators in whole serum, isolated serum exosomes, and exosome depleted serum were analyzed and compared between naïve and injured rats. Serum clinical chemistry analysis was performed to determine pathological changes. Results: In naïve animals, substantial amounts of IL-1β, IL-10, and TNF-α were encapsulated, IL-6 was completely encapsulated, and CXCL1 freely circulating. One hour after blast injury alone, levels of exosome encapsulated IFN-γ, IL-10, IL-6, IL-13, IL-4, and TNF-α increased, whereas freely circulating and membrane-associated levels remained undetectable or low. Rats with the most severe polytraumatic injuries with end organ complications had the earliest rise and most pronounced concentration of IL-1β, IL-10, TNF-α, and IL-6 across all serum compartments. Moreover, CXCL1 levels increased in relation to injury severity, but remained almost entirely freely circulating at all timepoints. Conclusion: These findings highlight that conventional ELISA-based assessments, which detect only free circulating and exosome membrane-bound mediators, underestimate the full immunoinflammatory response to trauma. Inclusion of exosome encapsulated mediators may be a better, more accurate and clinically useful early strategy to identify, diagnose, and monitor patients at highest risk for post-traumatic inflammation-associated complications.

Background: Traumatic heterotopic ossification (tHO) is characterized by ectopic bone formation in extra-skeletal sites leading to impaired wound healing, entrapment of neurovascular structures, pain, and reduced range of motion. HO has become a signature pathology affecting wounded military personnel who have sustained blast-associated traumatic amputations during the recent conflicts in Iraq and Afghanistan and can compound recovery by causing difficulty with prosthesis limb wearing. Tourniquet use to control catastrophic limb hemorrhage prior to surgery has become almost ubiquitous during this time, with the recognition the prolonged use may risk an ischemia reperfusion injury and associated complications. While many factors influence the formation of tHO, the extended use of tourniquets to limit catastrophic hemorrhage during prolonged field care has not been explored.Methods: Utilizing an established pre-clinical model of blast-associated complex lower limb injury and traumatic amputation, we evaluated the effects of tourniquet use on tHO formation. Adult male rats were subjected to blast overpressure exposure, femur fracture, and soft tissue crush injury. Pneumatic tourniquet (250–300 mmHg) applied proximal to the injured limb for 150-min was compared to a control group without tourniquet, before a trans-femoral amputation was performed. Outcome measures were volume to tHO formation at 12 weeks and changes in proteomic and genomic markers of early tHO formation between groups.Results: At 12 weeks, volumetric analysis with microCT imaging revealed a 70% increase in total bone formation (p = 0.007) near the site of injury compared to rats with no tourniquet time in the setting of blast-injuries. Rats subjected to tourniquet usage had increased expression of danger-associated molecular patterns (DAMPs) and end organ damage as early as 6 h and as late as 7 days post injury. The expressions of pro-inflammatory cytokines and chemokines and osteochondrogenic genes using quantitative RT-PCR similarly revealed increased expression as early as 6 h post injury, and these genes along with hypoxia associated genes remained elevated for 7 days compared to no tourniquet use.Conclusion: These findings suggest that tourniquet induced ischemia leads to significant increases in key transcription factors associated with early endochondral bone formation, systemic inflammatory and hypoxia, resulting in increased HO formation.

Trauma triggers critical molecular and cellular signaling cascades that drive biological outcomes and recovery. Variations in the gene expression of common endogenous reference housekeeping genes (HKGs) used in data normalization differ between tissue types and pathological states. Systematically, we investigated the gene stability of nine HKGs (Actb, B2m, Gapdh, Hprt1, Pgk1, Rplp0, Rplp2, Tbp, and Tfrc) from tissues prone to remote organ dysfunction (lung, liver, kidney, and muscle) following extremity trauma. Computational algorithms (geNorm, Normfinder, ΔCt, BestKeeper, RefFinder) were applied to estimate the expression stability of each HKG or combinations of them, within and between tissues, under both steady-state and systemic inflammatory conditions. Rplp2 was ranked as the most suitable in the healthy and injured lung, kidney, and skeletal muscle, whereas Rplp2 and either Hprt1 or Pgk1 were the most suitable in the healthy and injured liver, respectively. However, the geometric mean of the three most stable genes was deemed the most stable internal reference control. Actb and Tbp were the least stable in normal tissues, whereas Gapdh and Tbp were the least stable across all tissues post-trauma. Ct values correlated poorly with the translation from mRNA to protein. Our results provide a valuable resource for the accurate normalization of gene expression in trauma-related experiments.