Elevations in Circulating sST2 Levels Are Associated With In-Hospital Mortality and Adverse Clinical Outcomes After Blunt Trauma.

BACKGROUND: Soluble suppression of tumorigenicity 2 (sST2), a decoy receptor for interleukin (IL)-33, has emerged as a novel biomarker in various disease processes. Recent studies have elucidated the role of the sST2/IL-33 complex in modulating the balance of Th1/Th2 immune responses after tissue stress. However, the role of sST2 as a biomarker after traumatic injury remains unclear. To address this, we evaluated serum sST2 correlations with mortality and in-hospital adverse outcomes as endpoints in blunt trauma patients. METHODS: We retrospectively analyzed clinical and biobank data of 493 blunt trauma victims 472 survivors (mean age: 48.4 ± 0.87; injury severity score [ISS]: 19.6 ± 0.48) and 19 nonsurvivors (mean age: 58.8 ± 4.5; ISS: 23.3 ± 2.1) admitted to the intensive care unit. Given the confounding impact of age on the inflammatory response, we derived a propensity-matched survivor subgroup (n = 19; mean age: 59 ± 3; ISS: 23.4 ± 2) using an IBM SPSS case-control matching algorithm. Serial blood samples were obtained from all patients (3 samples within the first 24 h and then once daily from day [D] 1 to D5 after injury). sST2 and twenty-nine inflammatory biomarkers were assayed using enzyme-linked immunosorbent assay and Luminex, respectively. Two-way analysis of variance on ranks was used to compare groups (P < 0.05). Spearman rank correlation was performed to determine the association of circulating sST2 levels with biomarker levels and in-hospital clinical outcomes. RESULTS: Circulating sST2 levels of the nonsurvivor cohort were statistically significantly elevated at 12 h after injury and remained elevated up to D5 when compared either to the overall 472 survivor cohort or a matched 19 survivor subcohort. Admission sST2 levels obtained from the first blood draw after injury in the survivor cohort correlated positively with admission base deficit (correlation coefficient [CC] = 0.1; P = 0.02), international normalized ratio (CC = 0.1, P = 0.03), ISS (CC = 0.1, P = 0.008), and the average Marshall multiple organ dysfunction score between D2 and D5 (CC = 0.1, P = 0.04). Correlations with ISS revealed a positive correlation of ISS with plasma sST2 levels across the mild ISS (CC = 0.47, P < 0.001), moderate ISS (CC = 0.58, P < 0.001), and severe ISS groups (CC = 0.63, P < 0.001). Analysis of biomarker correlations in the matched survivor group over the initial 24 h after injury showed that sST2 correlates strongly and positively with IL-4 (CC = 0.65, P = 0.002), IL-5 (CC = 0.57, P = 0.01), IL-21 (CC = 0.52, P = 0.02), IL-2 (CC = 0.51, P = 0.02), soluble IL-2 receptor-α (CC = 0.5, P = 0.02), IL-13 (CC = 0.49, P = 0.02), and IL-17A (CC = 0.48, P = 0.03). This was not seen in the matched nonsurvivor group. sST2/IL-33 ratios were significantly elevated in nonsurvivors and patients with severe injury based on ISS ≥ 25. CONCLUSIONS: Elevations in serum sST2 levels are associated with poor clinical trajectories and mortality after blunt trauma. High sST2 coupled with low IL-33 associates with severe injury, mortality, and worse clinical outcomes. These findings suggest that sST2 could serve as an early prognostic biomarker in trauma patients and that sustained elevations of sST2 could contribute to a detrimental suppression of IL-33 bioavailability in patients with high injury severity.

IL33-mediated ILC2 activation and neutrophil IL5 production in the lung response after severe trauma: A reverse translation study from a human cohort to a mouse trauma model.

BACKGROUND: The immunosuppression and immune dysregulation that follows severe injury includes type 2 immune responses manifested by elevations in interleukin (IL) 4, IL5, and IL13 early after injury. We hypothesized that IL33, an alarmin released early after tissue injury and a known regulator of type 2 immunity, contributes to the early type 2 immune responses after systemic injury. METHODS AND FINDINGS: Blunt trauma patients admitted to the trauma intensive care unit of a level I trauma center were enrolled in an observational study that included frequent blood sampling. Dynamic changes in IL33 and soluble suppression of tumorigenicity 2 (sST2) levels were measured in the plasma and correlated with levels of the type 2 cytokines and nosocomial infection. Based on the observations in humans, mechanistic experiments were designed in a mouse model of resuscitated hemorrhagic shock and tissue trauma (HS/T). These experiments utilized wild-type C57BL/6 mice, IL33-/- mice, B6.C3(Cg)-Rorasg/sg mice deficient in group 2 innate lymphoid cells (ILC2), and C57BL/6 wild-type mice treated with anti-IL5 antibody. Severely injured human blunt trauma patients (n = 472, average injury severity score [ISS] = 20.2) exhibited elevations in plasma IL33 levels upon admission and over time that correlated positively with increases in IL4, IL5, and IL13 (P < 0.0001). sST2 levels also increased after injury but in a delayed manner compared with IL33. The increases in IL33 and sST2 were significantly greater in patients that developed nosocomial infection and organ dysfunction than similarly injured patients that did not (P < 0.05). Mechanistic studies were carried out in a mouse model of HS/T that recapitulated the early increase in IL33 and delayed increase in sST2 in the plasma (P < 0.005). These studies identified a pathway where IL33 induces ILC2 activation in the lung within hours of HS/T. ILC2 IL5 up-regulation induces further IL5 expression by CXCR2+ lung neutrophils, culminating in early lung injury. The major limitations of this study are the descriptive nature of the human study component and the impact of the potential differences between human and mouse immune responses to polytrauma. Also, the studies performed did not permit us to make conclusions about the impact of IL33 on pulmonary function. CONCLUSIONS: These results suggest that IL33 may initiate early detrimental type 2 immune responses after trauma through ILC2 regulation of neutrophil IL5 production. This IL33-ILC2-IL5-neutrophil axis defines a novel regulatory role for ILC2 in acute lung injury that could be targeted in trauma patients prone to early lung dysfunction.

Temporal Patterns of Circulating Inflammation Biomarker Networks Differentiate Susceptibility to Nosocomial Infection Following Blunt Trauma in Humans.

BACKGROUND: Severe traumatic injury can lead to immune dysfunction that renders trauma patients susceptible to nosocomial infections (NI) and prolonged intensive care unit (ICU) stays. We hypothesized that early circulating biomarker patterns following trauma would correlate with sustained immune dysregulation associated with NI and remote organ failure. METHODS: In a cohort of 472 blunt trauma survivors studied over an 8-year period, 127 patients (27%) were diagnosed with NI versus 345 trauma patients without NI. To perform a pairwise, case-control study with 1:1 matching, 44 of the NI patients were compared with 44 no-NI trauma patients selected by matching patient demographics and injury characteristics. Plasma obtained upon admission and over time were assayed for 26 inflammatory mediators and analyzed for the presence of dynamic networks. RESULTS: Significant differences in ICU length of stay (LOS), hospital LOS, and days on mechanical ventilation were observed in the NI patients versus no-NI patients. Although NI was not detected until day 7, multiple mediators were significantly elevated within the first 24 hours in patients who developed NI. Circulating inflammation biomarkers exhibited 4 distinct dynamic patterns, of which 2 clearly distinguish patients destined to develop NI from those who did not. Mediator network connectivity analysis revealed a higher, coordinated degree of activation of both innate and lymphoid pathways in the NI patients over the initial 24 hours. CONCLUSIONS: These studies implicate unique dynamic immune responses, reflected in circulating biomarkers that differentiate patients prone to persistent critical illness and infections following injury, independent of mechanism of injury, injury severity, age, or sex.

Computational Analysis Supports an Early, Type 17 Cell-Associated Divergence of Blunt Trauma Survival and Mortality.

OBJECTIVE: Blunt trauma patients may present with similar demographics and injury severity yet differ with regard to survival. We hypothesized that this divergence was due to different trajectories of systemic inflammation and utilized computational analyses to define these differences. DESIGN: Retrospective clinical study and experimental study in mice. SETTING: Level 1 trauma center and experimental laboratory. PATIENTS: From a cohort of 493 victims of blunt trauma, we conducted a pairwise, retrospective, case-control study of patients who survived over 24 hours but ultimately died (nonsurvivors; n = 19) and patients who, after ICU admission, went on to be discharged(survivors; n = 19). INTERVENTIONS: None in patients. Neutralizing anti-interleukin-17A antibody in mice. MEASUREMENTS AND MAIN RESULTS: Data on systemic inflammatory mediators assessed within the first 24 hours and over 7 days were analyzed with computational modeling to infer dynamic networks of inflammation. Network density among inflammatory mediators in nonsurvivors increased in parallel with organ dysfunction scores over 7 days, suggesting the presence of early, self-sustaining, pathologic inflammation involving high-mobility group protein B1, interleukin-23, and the Th17 pathway. Survivors demonstrated a pattern commensurate with a self-resolving, predominantly lymphoid response, including higher levels of the reparative cytokine interleukin-22. Mice subjected to trauma/hemorrhage exhibited reduced organ damage when treated with anti-interleukin-17A. CONCLUSIONS: Variable type 17 immune responses are hallmarks of organ damage, survival, and mortality after blunt trauma and suggest a lymphoid cell-based switch from self-resolving to self-sustaining inflammation.

Injury-induced MRP8/MRP14 stimulates IP-10/CXCL10 in monocytes/macrophages.

Trauma/hemorrhagic shock is associated with morbidity and mortality due to dysregulated inflammation, which is driven in part by monocytes/macrophages stimulated by injury-induced release of damage-associated molecular pattern (DAMP) molecules. MRP8/MRP14 is an endogenous DAMP involved in various inflammatory diseases, though its mechanism of action is unclear. Circulating MRP8/MRP14 levels in human blunt trauma nonsurvivors were significantly lower than those of survivors (P < 0.001). Human monocytic THP-1 cells stimulated with MRP8/MRP14 expressed the chemokine IFN-γ inducible protein 10 (IP-10)/CXCL10. Circulating IP-10 levels in human blunt trauma patients were correlated positively with MRP8/MRP14 levels (r = 0.396, P < 0.001), and were significantly lower in trauma nonsurvivors than in survivors (P < 0.001). We therefore sought to determine the mechanisms by which MRP8/MRP14 stimulates IP-10 in monocytes/macrophages, and found that induction of IP-10 by MRP8/MRP14 required Toll-like receptor 4 and TRIF but not MyD88. Full induction of IP-10 by MRP8/MRP14 required synergy between the transcription factors NF-κB and IFN regulatory factor 3 (IRF3). The receptor for IP-10 is CXCR3, and MRP8/MRP14-induced chemotaxis of CXCR3(+) cells was dependent on the production of IP-10 in monocytes/macrophages. Furthermore, in vivo study with a mouse trauma/hemorrhagic shock model showed that administration of neutralizing antibody against MRP8 prevented activation of NF-κB and IRF3 as well as IP-10 production. Thus, the current study identified a novel signaling mechanism that controls IP-10 expression in monocytes/macrophages by MRP8/MRP14, which may play an important role in injury-induced inflammation.

Elevated Admission Base Deficit Is Associated with a Complex Dynamic Network of Systemic Inflammation Which Drives Clinical Trajectories in Blunt Trauma Patients.

We hypothesized that elevated base deficit (BD) ≥ 4 mEq/L upon admission could be associated with an altered inflammatory response, which in turn may impact differential clinical trajectories. Using clinical and biobank data from 472 blunt trauma survivors, 154 patients were identified after excluding patients who received prehospital IV fluids or had alcohol intoxication. From this subcohort, 84 patients had a BD ≥ 4 mEq/L and 70 patients with BD < 4 mEq/L. Three samples within the first 24 h were obtained from all patients and then daily up to day 7 after injury. Twenty-two cytokines and chemokines were assayed using Luminex™ and were analyzed using two-way ANOVA and dynamic network analysis (DyNA). Multiple mediators of the innate and lymphoid immune responses in the BD ≥ 4 group were elevated differentially upon admission and up to 16 h after injury. DyNA revealed a higher, sustained degree of interconnectivity of the inflammatory response in the BD ≥ 4 patients during the initial 16 h after injury. These results suggest that elevated admission BD is associated with differential immune/inflammatory pathways, which subsequently could predispose patients to follow a complicated clinical course.

Prehospital Hypotension Is Associated With Altered Inflammation Dynamics and Worse Outcomes Following Blunt Trauma in Humans.

OBJECTIVE: To define the impact of prehospital hypotension on the dynamic, systemic acute inflammatory response to blunt trauma. DESIGN: Retrospective study. SETTINGS: Tertiary care institution. PATIENTS: Twenty-two hypotensive blunt trauma patients matched with 28 normotensive blunt trauma patients. INTERVENTION: None. MEASUREMENTS AND MAIN RESULTS: From a cohort of 472 blunt trauma survivors studied following institutional review board approval, two stringently matched subcohorts were derived. Twenty-two patients who sustained prehospital hypotension following blunt trauma (15 males and 7 females; age, 45 ± 3.8; Injury Severity Score, 20.7 ± 1.8) were matched with 28 normotensive trauma patients (20 males and 8 females; age, 46.1 ± 2.5; Injury Severity Score, 20.8 ± 1.3). Serial blood samples (three samples within the first 24 hr and then from days 1 to 7 postinjury) were assessed for 24 inflammatory mediators using Luminex, and No2-/No3- was measured using the nitrate reductase/Griess assay. Two-way analysis of variance was used to compare groups. Dynamic Bayesian Network inference was used to infer causal relationships based on probabilistic measures. Statistically significant differences were observed in ICU length of stay, total length of stay, days on mechanical ventilator, and Marshall Multiple Organ Dysfunction score between hypotensive and normotensive patients. Shock markers (shock index, pH, lactate, and base deficit) were significantly altered in hypotensive patients. Plasma levels of chemokines (monocyte chemotactic protein-1/CCL2, inducible protein-10/CXCL10, macrophage inflammatory protein-1α/CCL3, and interleukin-8/CCL8) and cytokines (interleukin-6, interleukin-10, interleukin-17, granulocyte-macrophage colony-stimulating factor, interleukin-1ß, and interleukin-7) as well as soluble interleukin-2 receptor-α were significantly elevated over the first 7 days postinjury in the hypotensive versus normotensive patients. Dynamic Bayesian Network suggested that the chemokines monocyte chemotactic protein-1/CCL2 and monokine induced by gamma interferon/CXCL9 in the hypotensive and normotensive patients, respectively, affect plasma interleukin-6 levels differentially in the initial 24 hours postinjury. CONCLUSIONS: Studies in stringently matched patient cohorts suggest that an episode of prehospital hypotension post trauma leads to early, dynamic reprogramming of systemic inflammation (including differential upstream regulation of interleukin-6), which is associated with worse outcomes.

X chromosome-linked IRAK-1 polymorphism is a strong predictor of multiple organ failure and mortality postinjury.

OBJECTIVE(S): Clinical research characterizing the mechanisms responsible for sex-based outcome differences postinjury remain conflicting. We sought to characterize an X chromosome-linked IRAK-1 (IL-1 receptor-associated kinase) polymorphism as an alternative mechanism responsible for sex differences postinjury. IRAK-1 is key intermediate in the toll-like receptor (TLR) pathway thought to drive inflammation postinjury. METHODS: A prospective cohort study was performed over a 24-month period. Bluntly injured patients requiring intensive care unit admission were enrolled, whereas patients with isolated brain and spinal cord injuries were excluded. Outcomes of interest included multiple organ failure (MOF, Marshall MOD score > 5) and mortality. Logistic regression was utilized to determine the independent risk of poor outcome associated with the IRAK-1 variant after controlling for important differences. RESULTS: In an enrolled cohort of 321 patients, the IRAK-1 variant was common (12.5%). Patients with and without the variant were similar in age, injury severity, and 24hr blood transfusion. After controlling for important confounders, the IRAK1 variant was independently associated with more than eightfold (OR = 8.4, P = 0.005, 95% CI: 1.9-37.1) and 11-fold (OR = 11.8, P = 0.037, 95% CI: 1.1-121) greater risk of MOF and mortality, respectively. These differences were most prominent in men, whereas women heterozygous for the variant demonstrated worse outcome in a dose-dependent fashion. CONCLUSIONS: The IRAK1 polymorphism is a strong independent predictor of MOF and mortality postinjury and represents a common variant with prognostic potential. These data demonstrate the importance of TLR signaling postinjury and supports that a genetic mechanism may drive sex outcome differences postinjury.

Inducible protein-10, a potential driver of neurally controlled interleukin-10 and morbidity in human blunt trauma.

OBJECTIVE: Blunt trauma and traumatic spinal cord injury induce systemic inflammation that contributes to morbidity. Dysregulated neural control of systemic inflammation postinjury is likely exaggerated in patients with traumatic spinal cord injury. We used in silico methods to discern dynamic inflammatory networks that could distinguish systemic inflammation in traumatic spinal cord injury from blunt trauma. DESIGN: Retrospective study. SETTINGS: Tertiary care institution. PATIENTS: Twenty-one severely injured thoracocervical traumatic spinal cord injury patients and matched 21 severely injured blunt trauma patients without spinal cord injury. INTERVENTION: None. MEASUREMENTS AND MAIN RESULTS: Serial blood samples were obtained from days 1 to 14 postinjury. Twenty-four plasma inflammatory mediators were quantified. Statistical significance between the two groups was determined by two-way analysis of variance. Dynamic Bayesian network inference was used to suggest dynamic connectivity and central inflammatory mediators. Circulating interleukin-10 was significantly elevated in thoracocervical traumatic spinal cord injury group versus non-spinal cord injury group, whereas interleukin-1ß, soluble interleukin-2 receptor-α, interleukin-4, interleukin-5, interleukin-7, interleukin-13, interleukin-17, macrophage inflammatory protein 1α and 1ß, granulocyte-macrophage colony-stimulating factor, and interferon-γ were significantly reduced in traumatic spinal cord injury group versus non-spinal cord injury group. Dynamic Bayesian network suggested that post-spinal cord injury interleukin-10 is driven by inducible protein-10, whereas monocyte chemotactic protein-1 was central in non-spinal cord injury dynamic networks. In a separate validation cohorts of 356 patients without spinal cord injury and 85 traumatic spinal cord injury patients, individuals with plasma inducible protein-10 levels more than or equal to 730 pg/mL had significantly prolonged hospital and ICU stay and days on mechanical ventilator versus patients with plasma inducible protein-10 level less than 730 pg/mL. CONCLUSION: This is the first study to compare the dynamic systemic inflammatory responses of traumatic spinal cord injury patients versus patients without spinal cord injury, suggesting a key role for inducible protein-10 in driving systemic interleukin-10 and morbidity and highlighting the potential utility of in silico tools to identify key inflammatory drivers.

Digital twin mathematical models suggest individualized hemorrhagic shock resuscitation strategies.

BACKGROUND: Optimizing resuscitation to reduce inflammation and organ dysfunction following human trauma-associated hemorrhagic shock is a major clinical hurdle. This is limited by the short duration of pre-clinical studies and the sparsity of early data in the clinical setting. METHODS: We sought to bridge this gap by linking preclinical data in a porcine model with clinical data from patients from the Prospective, Observational, Multicenter, Major Trauma Transfusion (PROMMTT) study via a three-compartment ordinary differential equation model of inflammation and coagulation. RESULTS: The mathematical model accurately predicts physiologic, inflammatory, and laboratory measures in both the porcine model and patients, as well as the outcome and time of death in the PROMMTT cohort. Model simulation suggests that resuscitation with plasma and red blood cells outperformed resuscitation with crystalloid or plasma alone, and that earlier plasma resuscitation reduced injury severity and increased survival time. CONCLUSIONS: This workflow may serve as a translational bridge from pre-clinical to clinical studies in trauma-associated hemorrhagic shock and other complex disease settings.

Research to improve survival in patients with severe bleeding after major trauma presents many challenges. Here, we created a computer model to simulate the effects of severe bleeding. We refined this model using data from existing animal studies to ensure our simulations were accurate. We also used patient data to further refine the simulations to accurately predict which patients would live and which would not. We studied the effects of different treatment protocols on these simulated patients and show that treatment with plasma (the fluid portion of blood that helps form blood clots) and red blood cells jointly, gave better results than treatment with intravenous fluid or plasma alone. Early treatment with plasma reduced injury severity and increased survival time. This modelling approach may improve our ability to evaluate new treatments for trauma-associated bleeding and other acute conditions.

Identification of a novel pathway of transforming growth factor-ß1 regulation by extracellular NAD+ in mouse macrophages: in vitro and in silico studies.

Extracellular ß-nicotinamide adenine dinucleotide (NAD(+)) is anti-inflammatory. We hypothesized that NAD(+) would modulate the anti-inflammatory cytokine Transforming Growth Factor (TGF)-ß1. Indeed, NAD(+) led to increases in both active and latent cell-associated TGF-ß1 in RAW 264.7 mouse macrophages as well as in primary peritoneal macrophages isolated from both C3H/HeJ (TLR4-mutant) and C3H/HeOuJ (wild-type controls for C3H/HeJ) mice. NAD(+) acts partially via cyclic ADP-ribose (cADPR) and subsequent release of Ca(2+). Treatment of macrophages with the cADPR analog 3-deaza-cADPR or Ca(2+) ionophores recapitulated the effects of NAD(+) on TGF-ß1, whereas the cADPR antagonist 8-Br-cADPR, Ca(2+) chelation, and antagonism of L-type Ca(2+) channels suppressed these effects. The time and dose effects of NAD(+) on TGF-ß1 were complex and could be modeled both statistically and mathematically. Model-predicted levels of TGF-ß1 protein and mRNA were largely confirmed experimentally but also suggested the presence of other mechanisms of regulation of TGF-ß1 by NAD(+). Thus, in vitro and in silico evidence points to NAD(+) as a novel modulator of TGF-ß1.

A common single nucleotide polymorphism is associated with inflammation and critical illness outcomes.

Acute inflammation is heterogeneous in critical illness and predictive of outcome. We hypothesized that genetic variability in novel, yet common, gene variants contributes to this heterogeneity and could stratify patient outcomes. We searched algorithmically for significant differences in systemic inflammatory mediators associated with any of 551,839 SNPs in one derivation (n = 380 patients with blunt trauma) and two validation (n = 75 trauma and n = 537 non-trauma patients) cohorts. This analysis identified rs10404939 in the LYPD4 gene. Trauma patients homozygous for the A allele (rs10404939AA; 27%) had different trajectories of systemic inflammation along with persistently elevated multiple organ dysfunction (MOD) indices vs. patients homozygous for the G allele (rs10404939GG; 26%). rs10404939AA homozygotes in the trauma validation cohort had elevated MOD indices, and non-trauma patients displayed more complex inflammatory networks and worse 90-day survival compared to rs10404939GG homozygotes. Thus, rs10404939 emerged as a common, broadly prognostic SNP in critical illness.

Hemoadsorption reprograms inflammation in experimental gram-negative septic peritonitis: insights from in vivo and in silico studies.

UNLABELLED: Improper compartmentalization of the inflammatory response leads to systemic inflammation in sepsis. Hemoadsorption (HA) is an emerging approach to modulate sepsis-induced inflammation. We sought to define the effects of HA on inflammatory compartmentalization in Escherichia coli-induced fibrin peritonitis in rats. HYPOTHESIS: HA both reprograms and recompartmentalizes inflammation in sepsis. Sprague Dawley male rats were subjected to E. coli peritonitis and, after 24 h, were randomized to HA or sham treatment (sepsis alone). Venous blood samples collected at 0, 1, 3 and 6 h (that is, 24-30 h of total experimental sepsis), and peritoneal samples collected at 0 and 6 h, were assayed for 14 cytokines along with NO(2)(-/)NO(3)(-). Bacterial counts were assessed in the peritoneal fluid at 0 and 6 h. Plasma tumor necrosis factor (TNF)-α, interleukin (IL)-6, CXCL-1, and CCL2 were significantly reduced in HA versus sham. Principal component analysis (PCA) suggested that inflammation in sham was driven by IL-6 and TNF-α, whereas HA-associated inflammation was driven primarily by TNF-α, CXCL-1, IL-10 and CCL2. Whereas -peritoneal bacterial counts, plasma aspartate transaminase levels and peritoneal IL-5, IL-6, IL-18, interferon (IFN)-γ and NO(2)(-)/NO(3)(-) were significantly lower, both CXCL-1 and CCL2 as well as the peritoneal-to-plasma ratios of TNF-α, CXCL-1 and CCL2 were significantly higher in HA versus sham, suggesting that HA-induced inflammatory recompartmentalization leads to the different inflammatory drivers discerned in part by PCA. In conclusion, this study demonstrates the utility of combined in vivo/in silico methods and suggests that HA exerts differential effects on mediator gradients between local and systemic compartments that ultimately benefit the host.

Sepsis: from pattern to mechanism and back.

Sepsis is a clinical entity in which complex inflammatory and physiological processes are mobilized, not only across a range of cellular and molecular interactions, but also in clinically relevant physiological signals accessible at the bedside. There is a need for a mechanistic understanding that links the clinical phenomenon of physiologic variability with the underlying patterns of the biology of inflammation, and we assert that this can be facilitated through the use of dynamic mathematical and computational modeling. An iterative approach of laboratory experimentation and mathematical/computational modeling has the potential to integrate cellular biology, physiology, control theory, and systems engineering across biological scales, yielding insights into the control structures that govern mechanisms by which phenomena, detected as biological patterns, are produced. This approach can represent hypotheses in the formal language of mathematics and computation, and link behaviors that cross scales and domains, thereby offering the opportunity to better explain, diagnose, and intervene in the care of the septic patient.

The independent prognostic value of global epigenetic alterations: An analysis of single-cell ATAC-seq of circulating leukocytes from trauma patients followed by validation in whole blood leukocyte transcriptomes across three etiologies of critical illness.

BACKGROUND: While bulk and single cell transcriptomic patterns in circulating leukocytes from trauma patients have been reported, how these relate to changes in open chromatin patterns remain unstudied. Here, we investigated whether single-cell ATAC-seq would provide further resolution of transcriptomic patterns that align with patient outcomes. METHODS: We performed scATAC-seq on peripheral blood mononuclear cells from four trauma patients at <4 h, 24 h, 72 h post-injury and four matched healthy controls, and extracted the features associated with the global epigenetic alterations. Three large-scale bulk transcriptomic datasets from trauma, burn and sepsis patients were used to validate the scATAC-seq derived signature, explore patient epigenetic heterogeneity (Epigenetic Groups: EG_hi vs. EG_lo), and associate patterns with clinical outcomes in critical illness. FINDINGS: Patient subsets with gene expression patterns in blood leukocytes representative of a high global epigenetic signature (EG_hi) had worse outcomes across three etiologies of critical illness. EG_hi designation contributed independent of the known immune leukocyte transcriptomic responses to patient prognosis (Trauma: HR=0.62 [95% CI: 0.43-0.89, event set as recovery], p=0.01, n=167; Burns: HR=4.35 [95% CI: 0.816-23.2, event set as death], p=0.085, n=121; Sepsis: HR=1.60 [95% CI: 1.10-2.33, event set as death], p=0.013, n=479; Cox proportional hazards regression). INTERPRETATION: The inclusion of gene expression patterns that associate with global epigenetic changes in circulating leukocytes improves the resolution of transcriptome-based patient classification in acute critical illnesses. Early detection of both the global epigenetic signature and the known immune transcriptomic patterns associates with the worse prognosis in trauma, burns and sepsis. FUNDING: This project was supported by an R35 grant from National Institutes of Health: 1R35GM127027-01 (T.B.).

Plasma proteomics reveals early, broad release of chemokine, cytokine, TNF, and interferon mediators following trauma with delayed increases in a subset of chemokines and cytokines in patients that remain critically ill.

Severe injury is known to cause a systemic cytokine storm that is associated with adverse outcomes. However, a comprehensive assessment of the time-dependent changes in circulating levels of a broad spectrum of protein immune mediators and soluble immune mediator receptors in severely injured trauma patients remains uncharacterized. To address this knowledge gap, we defined the temporal and outcome-based patterns of 184 known immune mediators and soluble cytokine receptors in the circulation of severely injured patients. Proteomics (aptamer-based assay, SomaLogic, Inc) was performed on plasma samples drawn at 0, 24, and 72 hours (h) from time of admission from 150 trauma patients, a representative subset from the Prehospital Plasma during Air Medical Transport in Trauma Patients at Risk for Hemorrhagic Shock (PAMPer) trial. Patients were categorized into outcome groups including Early Non-Survivors (died within 72 h; ENS; n=38), Non-Resolvers (died after 72 h or required ≥7 days of intensive care; NR; n=78), and Resolvers (survivors that required < 7 days of intensive care; R; n=34), with low Injury Severity Score (ISS) patients from the Tranexamic Acid During Prehospital Transport in Patients at Risk for Hemorrhage After Injury (STAAMP) trial as controls. The major findings include an extensive release of immune mediators and cytokine receptors at time 0h that is more pronounced in ENS and NR patients. There was a selective subset of mediators elevated at 24 and 72 h to a greater degree in NR patients, including multiple cytokines and chemokines not previously described in trauma patients. These findings were validated in a quantitative fashion using mesoscale discovery immunoassays (MSD) from an external validation cohort (VC) of samples from 58 trauma patients matched for R and NR status. This comprehensive longitudinal description of immune mediator patterns associated with trauma outcomes provides a new level of characterization of the immune response that follows severe injury.

Lipidomic signatures align with inflammatory patterns and outcomes in critical illness.

Alterations in lipid metabolism have the potential to be markers as well as drivers of pathobiology of acute critical illness. Here, we took advantage of the temporal precision offered by trauma as a common cause of critical illness to identify the dynamic patterns in the circulating lipidome in critically ill humans. The major findings include an early loss of all classes of circulating lipids followed by a delayed and selective lipogenesis in patients destined to remain critically ill. The previously reported survival benefit of early thawed plasma administration was associated with preserved lipid levels that related to favorable changes in coagulation and inflammation biomarkers in causal modelling. Phosphatidylethanolamines (PE) were elevated in patients with persistent critical illness and PE levels were prognostic for worse outcomes not only in trauma but also severe COVID-19 patients. Here we show selective rise in systemic PE as a common prognostic feature of critical illness.

The Use of Multiplexing to Identify Cytokine and Chemokine Networks in the Immune-Inflammatory Response to Trauma.

Significance: The immunoinflammatory responses that follow trauma contribute to clinical trajectory and patient outcomes. While remarkable advances have been made in trauma services and injury management, clarity on how the immune system in humans responds to trauma is lagging. Recent Advances: Multiplexing platforms have transformed our ability to analyze comprehensive immune mediator responses in human trauma. In parallel, with the establishment of large data sets, computational methods have been adapted to yield new insights based on mediator patterns. These efforts have added an important data layer to the emerging multiomic characterization of the human response to injury. Critical Issues: Outcome after trauma is greatly affected by the host immunoinflammatory response. Excessive or sustained responses can contribute to organ damage. Hence, understanding the pathophysiology behind traumatic injury is of vital importance. Future Directions: This review summarizes our work in the study of circulating immune mediators in trauma patients. Our foundational studies into dynamic patterns of inflammatory mediators represent an important contribution to the concepts and computational challenges that these large data sets present. We hope to see further integration and understanding of multiomics strategies in the field of trauma that can aid in patient endotyping and in potentially identifiying certain therapeutic targets in the future. Antioxid. Redox Signal. 35, 1393-1406.

Protective/reparative cytokines are suppressed at high injury severity in human trauma.

BACKGROUND: Trauma elicits a complex inflammatory response that, among multiple presenting factors, is greatly impacted by the magnitude of injury severity. Herein, we compared the changes in circulating levels of mediators with known proinflammatory roles to those with known protective/reparative actions as a function of injury severity in injured humans. METHODS: Clinical and biobank data were obtained from 472 (trauma database-1 (TD-1), University of Pittsburgh) and 89 (trauma database-2 (TD-2), Indiana University) trauma patients admitted to the intensive care unit (ICU) and who survived to discharge. Injury severity was estimated based on the Injury Severity Score (ISS), and this was used as both a continuous variable and for the purpose of grouping patients into severity-based cohorts. Samples within the first 24 hours were obtained from all patients and then daily up to day 7 postinjury in TD-1. Sixteen cytokines were assayed using Luminex and were analyzed using two-way analysis of variance (p<0.05). RESULTS: Patients with higher ISSs had longer ICU and hospital stays, days on mechanical ventilation and higher rates of nosocomial infection when compared with the mild and moderate groups. Time course analysis and correlations with ISS showed that 11 inflammatory mediators correlated positively with injury severity, consistent with previous reports. However, five mediators (interleukin (IL)-9, IL-21, IL-22, IL-23 and IL-17E/25) were suppressed in patients with high ISS and inversely correlated with ISS. DISCUSSION: These findings suggest that severe injury is associated with a suppression of a subset of cytokines known to be involved in tissue protection and regeneration (IL-9, IL-22 and IL-17E/25) and lymphocyte differentiation (IL-21 and IL-23), which in turn correlates with adverse clinical outcomes. Thus, patterns of proinflammatory versus protective/reparative mediators diverge with increasing ISS.

Early dynamic orchestration of immunologic mediators identifies multiply injured patients who are tolerant or sensitive to hemorrhage.

BACKGROUND: Multiply injured patients (MIPs) are at risk of complications including infections, and acute and prolonged organ dysfunction. The immunologic response to injury has been shown to affect outcomes. Recent advances in computational capabilities have shown that early dynamic coordination of the immunologic response is associated with improved outcomes after trauma. We hypothesized that patients who were sensitive or tolerant of hemorrhage would demonstrate differences in dynamic immunologic orchestration within hours of injury. METHODS: We identified two groups of MIPs who demonstrated distinct clinical tolerance to hemorrhage (n = 10) or distinct clinical sensitivity to hemorrhage (n = 9) from a consecutive cohort of 100 MIPs. Hemorrhage was quantified by integrating elevated shock index values for 24 hours after injury (shock volume). Clinical outcomes were quantified by average Marshall Organ Dysfunction Scores from days 2 to 5 after injury. Shock-sensitive patients had high cumulative organ dysfunction after lower magnitude hemorrhage. Shock-tolerant (ST) patients had low cumulative organ dysfunction after higher magnitude hemorrhage. Computational methods were used to analyze a panel of 20 immunologic mediators collected serially over the initial 72 hours after injury. RESULTS: Dynamic network analysis demonstrated the ST patients had increased orchestration of cytokines that are reparative and protective including interleukins 9, 17E/25, 21, 22, 23, and 33 during the initial 0- to 8-hour and 8- to 24-hour intervals after injury. Shock-sensitive patients had delayed immunologic orchestration of a network of largely proinflammatory and anti-inflammatory mediators. Elastic net linear regression demonstrated that a group of five mediators could discriminate between shock-sensitive and ST patients. CONCLUSIONS: Preliminary evidence from this study suggests that early immunologic orchestration discriminates between patients who are notably tolerant or sensitive to hemorrhage. Early orchestration of a group of reparative/protective mediators was amplified in shock-tolerant patients. LEVEL OF EVIDENCE: Prospective clinical outcomes study, level III.