Propranolol Normalizes Metabolomic Signatures Thereby Improving Outcomes After Burn.

OBJECTIVE AND BACKGROUND: Propranolol, a nonselective beta-receptor blocker, improves outcomes of severely burned patients. While the clinical and physiological benefits of beta-blockade are well characterized, the underlying metabolic mechanisms are less well defined. We hypothesized that propranolol improves outcomes after burn injury by profoundly modulating metabolic pathways. METHODS: In this phase II randomized controlled trial, patients with burns ≥20% of total body surface area were randomly assigned to control or propranolol (dose given to decrease heart rate <100 bpm). Outcomes included clinical markers, inflammatory and lipidomic profiles, untargeted metabolomics, and molecular pathways. RESULTS: Fifty-two severely burned patients were enrolled in this trial (propranolol, n=23 and controls, n=29). There were no significant differences in demographics or injury severity between groups. Metabolomic pathway analyses of the adipose tissue showed that propranolol substantially alters several essential metabolic pathways involved in energy and nucleotide metabolism, as well as catecholamine degradation ( P <0.05). Lipidomic analysis revealed that propranolol-treated patients had lower levels of proinflammatory palmitic acid ( P <0.05) and saturated fatty acids ( P <0.05) with an increased ratio of polyunsaturated fatty acids ( P <0.05), thus shifting the lipidomic profile towards an anti-inflammatory phenotype after burn ( P <0.05). These metabolic effects were mediated by decreased activation of hormone-sensitive lipase at serine 660 ( P <0.05) and significantly reduced endoplasmic reticulum stress by decreasing phospho-JNK ( P <0.05). CONCLUSION: Propranolol's ability to mitigate pathophysiological changes to essential metabolic pathways results in significantly improved stress responses.

NLRP3 knockout enhances immune infiltration and inflammatory responses and improves survival in a burn sepsis model.

Although sepsis in burn patients is a major contributor to mortality, treatments are not always effective and underlying mechanisms have yet to be completely elucidated. NLRP3 inflammasome orchestrates burn-induced, inflammatory-driven pathophysiologic processes. Here, we determined the mechanism of NLRP3 inflammasome activation on bacterial clearance and mortality in burn sepsis. We obtained tissue and blood from 30 wild-type and 30 Nlrp3-/- mice. Mice were subjected to a two-hit model of 25-30% TBSA scald burn followed by Pseudomonas aeruginosa wound infection 72 hours after injury. We also obtained tissue from 34 adult burn patients (≥18 years of age) with early (0-11 days post-burn) and later (≥12 days post-burn) surgical time-points and ten healthy controls. Murine studies indicated that Nlrp3-/- had 30% improved survival and bacterial clearance at the site of injury and is systemically relative to burn sepsis wild type. Greater macrophage and neutrophil infiltration occurred acutely after infection (12 hours) to the site of injury and adipose tissue. This was followed by increased macrophage and neutrophil infiltration to lymphoid organs and liver beyond the acute phase (24 and 72 hours). Interestingly, Nlrp3 ablation increased acute systemic inflammation (IL-6, TNF-α, IL-1ß). Septic burn patients had persistently increased adipose NLRP3 by-product expression beyond the acute phase that was more pronounced in late-onset sepsis. Our findings suggest that Nlrp3 genetic ablation enhanced acute tissue-specific inflammatory responsiveness. Likely, this occurs by paradoxically increasing acute immune infiltration and inflammation with a non-persistent response. Clinically, persistent NLRP3-mediated inflammation occurs in septic versus normal burn patients and potentially detrimentally impacts patient outcomes.

Alternatively Activated Macrophages Drive Browning of White Adipose Tissue in Burns.

OBJECTIVE: The aim of this study was to uncover the mediators and mechanistic events that facilitate the browning of white adipose tissue (WAT) in response to burns. BACKGROUND: In hypermetabolic patients (eg, burns, cancer), the browning of WAT has presented substantial clinical challenges related to cachexia, atherosclerosis, and poor clinical outcomes. Browning of the adipose tissue has recently been found to induce and sustain hypermetabolism. Although browning appears central in trauma-, burn-, or cancer-induced hypermetabolic catabolism, the mediators are essentially unknown. METHODS: WAT and blood samples were collected from patients admitted to the Ross Tilley Burn Centre at Sunnybrook Hospital. Wild type, CCR2 KO, and interleukin (IL)-6 KO male mice were purchased from Jax laboratories and subjected to a 30% total body surface area burn injury. WAT and serum collected were analyzed for browning markers, macrophages, and metabolic state via histology, gene expression, and mitochondrial respiration. RESULTS: In the present study, we show that burn-induced browning is associated with an increased macrophage infiltration, with a greater type 2 macrophage profile in the fat of burn patients. Similar to our clinical findings in burn patients, both an increase in macrophage recruitment and a type 2 macrophage profile were also observed in post burn mice. Genetic loss of the chemokine CCR2 responsible for macrophage migration to the adipose impairs burn-induced browning. Mechanistically, we show that macrophages recruited to burn-stressed subcutaneous WAT (sWAT) undergo alternative activation to induce tyrosine hydroxylase expression and catecholamine production mediated by IL-6, factors required for browning of sWAT. CONCLUSION: Together, our findings uncover macrophages as the key instigators and missing link in trauma-induced browning.

Acute Phase Response in Critically Ill Elderly Burn Patients.

OBJECTIVES: Survival of elderly burn patients remains unacceptably poor. The acute phase, defined as the first 96 hours after burn, includes the resuscitation period and influences subsequent outcomes and survival. The aim of this study was to determine if the acute phase response post burn injury is significantly different in elderly patients compared with adult patients and to identify elements contributing to adverse outcomes. DESIGN: Cohort study. SETTING: Tertiary burn center. PATIENTS: Adult (< 65 yr old) and elderly (≥ 65 yr old) patients with an acute burn injury. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: We included all patients with an acute burn injury greater than or equal to 20% total body surface area to our burn center from 2011 to 2016. Clinical and laboratory measures during the acute phase were compared between adult and elderly patients. Outcomes included clinical hemodynamic measurements, organ biomarkers, volume of fluid resuscitation, cardiac agents, and the inflammatory cytokine response in plasma. Data were analyzed using the Student t test, Mann-Whitney U test, and Fisher exact test. A total of 149 patients were included, with 126 adults and 23 elderly. Injury severity was not significantly different among adult and elderly patients. Elderly had significantly lower heart rates (p < 0.05), cardiac index (p < 0.05), mean arterial pressure (p < 0.05), PaO2/FIO2 (p < 0.05), and pH (p < 0.05), along with higher lactate (p < 0.05). Organ biomarkers, particularly creatinine and blood urea nitrogen, showed distinct differences between adults and elderly (p < 0.05). Elderly had significantly lower levels of interleukin-6, monocyte chemotactic protein-1, monocyte chemotactic protein-3, and granulocyte-colony stimulating factor during the acute phase (p < 0.05). Overall mortality was significantly higher in elderly patients (5% vs 52%; p < 0.0001). CONCLUSIONS: Response to the burn injury during the acute phase response after burn is substantially different between elderly and adult burn patients and is characterized by cardiac depression and hypoinflammation.

Pathophysiological Response to Burn Injury in Adults.

OBJECTIVE: The aim of this study was to compare the hypermetabolic, and inflammatory trajectories in burned adults to gain insight into the pathophysiological alterations and outcomes after injury. SUMMARY OF BACKGROUND DATA: Burn injury leads to a complex response that is associated with hypermetabolism, morbidity, and mortality. The underlying pathophysiology and the correlations between humoral changes and organ function have not been well delineated in adult burn patients. METHODS: Burned adult patients (n = 1288) admitted to our center from 2006 to 2016 were enrolled in this prospective study. Demographics, clinical data, metabolic and inflammatory markers, hypermetabolism, organ function, and clinical outcomes were obtained throughout acute hospitalization. We then stratified patients according to burn size (<20%, 20% to 40%, and >40% total body surface area [TBSA]) and compared biomedical profiles and clinical outcomes for these patients. RESULTS: Burn patients were hypermetabolic with elevated resting energy expenditure (REE) associated with increased browning of white adipose tissue from weeks 2 to 4. Hyperglycemia and hyperinsulinemia peaked 7 to 14 days after injury. Oral glucose tolerance and insulin resistance (QUICKI, HOMA2) tests further confirmed these findings with similar areas under the curve for moderate (20% to 40% TBSA) and severe burn (>40% TBSA). Lipid metabolism in sera revealed elevated pro-inflammatory stearic and linoleic acid, with complementary increases in anti-inflammatory free fatty acids. Similar increases were observed for inflammatory cytokines, chemokines, and metabolic hormones. White adipose tissue from the site of injury had increased ER stress, mitochondrial damage, and inflammasome activity, which was exacerbated with increasing burn severity. CONCLUSIONS: In this large prospective trial, we delineated the complexity of the pathophysiologic responses postburn in adults and concluded that these profound responses are time and burn size dependent. Patients with medium-size (20% to 40% TBSA) burn demonstrated a very robust response that is similar to large burns.

Induction of antigen-specific TH 9 immunity accompanied by mast cell activation blocks tumor cell engraftment.

The engraftment of circulating cancer cells at distal sites represents a key step in the metastatic cascade, yet remains an unexplored target for therapeutic intervention. In this study, we establish that a vaccination strategy yielding an antigen-specific TH 9 response induces long term host surveillance and prevents the engraftment of circulating cancer cells. Specifically, we show that vaccination with a recombinant CEA IgV-like N domain, formulated with the TLR3 ligand poly I:C, elicits a CEA-specific TH 9 response, wherein IL-9 secreting TH cells act in concert with CEA N domain-specific antibodies as well as activated mast cells in preventing tumor cell engraftment. The development of this immune response was dependent on TLR3, since interference with the TLR3-dsRNA complex formation led to a reduction in vaccine-imparted protection and a shift in the resulting immune response toward a TH 2 response. These findings point to the existence of an alternate tumor targeting immune mechanism that can be exploited for the purpose of developing vaccine therapies targeting tumor dissemination and engraftment.

Impaired Immune Response in Elderly Burn Patients: New Insights Into the Immune-senescence Phenotype.

OBJECTIVE: Comparing the inflammatory and immunological trajectories in burned adults versus burned elderly patients to gain novel insights and better understanding why elderly have poor outcomes. SUMMARY BACKGROUND DATA: Despite receiving the same treatment and clinical consideration as all other burn patients, elderly patients continue to have substantially poorer outcomes compared with adults. In light of an aging population, gaining a better understanding of their susceptibility to complications and creating new treatment strategies is imperative. METHODS: We included 130 burn patients (94 adults: <65 years old and 36 elderly: ≥65 years old) and 10 healthy controls in this study. Immune activity and expression was assessed using bioplex at various time points. Clinical outcomes such as infection, sepsis, and mortality were prospectively collected. RESULTS: Elderly burn patients had significantly lower burn size but significantly higher Baux scores. Morbidity and mortality was significantly increased in the elderly cohort. Immune biomarkers indicated that elderly are immune compromised and unable to respond with the expected inflammatory response during the early phase after injury. This trajectory changes to a hyperinflammatory pattern during the later phase after burn. These findings are even more pronounced when comparing sepsis versus nonsepsis patients as well as survivors versus nonsurvivors in the elderly. CONCLUSIONS: Elderly burned patients mount a delayed immune and dampened inflammatory response early after burn injury that changes to an augmented response at later time points. Late-onset sepsis and nonsurvivors had an immune exhaustion phenotype, which may represent one of the main mediators responsible for the striking mortality in elderly.

Glucose Control in Severely Burned Patients Using Metformin: An Interim Safety and Efficacy Analysis of a Phase II Randomized Controlled Trial.

OBJECTIVE: To determine whether metformin can achieve glucose control no worse than insulin (noninferiority) without the danger of hypoglycemia (superiority). In addition, to assess whether metformin has any additional effects on lipolysis and inflammation that will enhance burn recovery (superiority). SUMMARY BACKGROUND DATA: Hyperglycemia and insulin resistance after burn injury are associated with increased morbidity and mortality. Insulin administration improves postburn infections, severity of sepsis, and morbidity, but also causes a 4-5-fold increase in hypoglycemia, which is associated with a 9-fold increase in mortality. METHODS: Severely burned adult patients with burns over 20% total body surface area (TBSA) burn were prospectively randomized in this Phase II clinical trial to either metformin or insulin (standard of care) treatment. Primary outcomes were glucose levels and incidence of hypoglycemia. Secondary outcomes included glucose and fat metabolism, and clinical outcomes. RESULTS: Forty-four patients were enrolled in this Phase II clinical trial, 18 metformin and 26 insulin patients. Demographics, burn size, concomitant injuries, and mortality were comparable between both groups. Metformin controlled blood glucose as equally as insulin with no difference between the 2 treatment groups, P > 0.05. While there was a 15% incidence of hypoglycemia in the insulin group, there was only 1 mild hypoglycemic episode (6%) in the metformin group, P < 0.05. Oral glucose tolerance tests at discharge revealed that metformin significantly improved insulin sensitivity, P < 0.05. Furthermore, metformin had a strong antilipolytic effect after burn injury when compared with insulin and was associated with significantly reduced inflammation, P < 0.05. CONCLUSIONS: Metformin decreases glucose equally as effective as insulin without causing hypoglycemia, with additional benefits including improved insulin resistance and decreased endogenous insulin synthesis when compared with insulin controls. These results indicate that metformin is safe in burn patients and further supports the use of metformin in severely burned patients for postburn control of hyperglycemia and insulin resistance.

C-C Chemokine Receptor Type 2 Expression on Monocytes Before Sepsis Onset Is Higher Than That of Postsepsis in Septic Burned Patients: A New Predictor for Sepsis in Burned Injury.

OBJECTIVE: The present study aims to investigate the alterations in monocytes (Mo) and dendritic cells (DCs) in septic burned patients with a special focus on C-C chemokine receptor type 2 (CCR2) expressions on classical Mo. BACKGROUND: The phenotypes of Mo and DCs, particularly CCR2 expression on Mo, are not fully explored in severely burned patients with sepsis. METHODS: The prospective cohort study was conducted in Ross Tilley Burn Centre and Sunnybrook Research Institute (Toronto, Canada). We enrolled 8 healthy patients and 89 burned patients with various burned sizes, of those burned patients, 12 were with sepsis. Blood was collected upon admission to the hospital and throughout their course in hospital. The expression of human leukocyte antigen-DR was determined on all DCs and Mo, along with CCR2 on CD14/CD16 Mo. RESULTS: We found a profound decrease in human leukocyte antigen-DR on Mo and DCs in burned patients with sepsis compared with healthy controls and nonseptic burned patients. In addition, septic burned patients presented an increased CCR2 expression on classical Mo (CD14/CD16), which was paralleled by greater chemokine (C-C motif) ligand 2 concentrations in the plasma when compared with controls and nonseptic burned patients. Furthermore, burned patients with sepsis had a more profound expansion of CD14/CD16 Mo when compared with nonseptic burned patients. CONCLUSION: Our results demonstrate that burned patients with sepsis have more profound impairment of monocytes and dendritic cells than burned patients without sepsis. With CCR2 level on Mo before sepsis onset being higher than postsepsis, CCR2 expression could be a new predictor of sepsis onset in severe burn injury.

Anabolic and anticatabolic agents in critical care.

PURPOSE OF REVIEW: A complex network of hormones and other effectors characterize the hypermetabolic response in critical illness; these mediators work together to induce numerous pathophysiologic alterations. Increased incidence of infection, multiorgan failure, long-term debilitation, delays in rehabilitation, and death result from an inability to meet the prohibitively elevated protein and energy requirements, which occur during illness and can persist for several years. Pharmacologic interventions have been successfully utilized to attenuate particular aspects of the hypermetabolic response; these modalities are a component of managing critically ill patients - including those patients with severe burns. Here, we review recent advances in pharmacologically attenuating the hypermetabolic and catabolic responses. RECENT FINDINGS: Propranolol, a nonspecific ß-adrenergic receptor antagonist, is one of the most widely used anticatabolic therapies. Oxandrolone, testosterone, and intensive insulin therapy represent anabolic pharmacological strategies. Promising therapies, such as metformin, glucagon-like peptide 1, peroxisome proliferator-activated receptor agonists, are currently being investigated. SUMMARY: Profound metabolic derangements occur in critically ill patients; this hypermetabolic response is a major contributor to adverse outcomes. Despite the pharmacological therapies currently available to counteract this devastating cascade, future studies are warranted to explore new multimodality agents that will counteract these effects while maintaining glycemic control and preventing unfavorable complications.

Leukocyte infiltration and activation of the NLRP3 inflammasome in white adipose tissue following thermal injury.

OBJECTIVES: Severe thermal injury is associated with extreme and prolonged inflammatory and hypermetabolic responses, resulting in significant catabolism that delays recovery or even leads to multiple organ failure and death. Burned patients exhibit many symptoms of stress-induced diabetes, including hyperglycemia, hyperinsulinemia, and hyperlipidemia. Recently, the nucleotide-binding domain, leucine-rich family (NLR), pyrin-containing 3 (NLRP3) inflammasome has received much attention as the sensor of endogenous "danger signals" and mediator of "sterile inflammation" in type II diabetes. Therefore, we investigated whether the NLRP3 inflammasome is activated in the adipose tissue of burned patients, as we hypothesize that, similar to the scenario observed in chronic diabetes, the cytokines produced by the inflammasome mediate insulin resistance and metabolic dysfunction. DESIGN: Prospective cohort study. SETTING: Ross Tilley Burn Centre & Sunnybrook Research Institute. PATIENTS: We enrolled 76 patients with burn sizes ranging from 1% to 70% total body surface area. All severely burned patients exhibited burn-induced insulin resistance and hyperglycemia. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: We examined the adipose tissue of control and burned patients and found, via flow cytometry and gene expression studies, increased infiltration of leukocytes-especially macrophages-and evidence of inflammasome priming and activation. Furthermore, we observed increased levels of interleukin-1ß in the plasma of burned patients when compared to controls. CONCLUSIONS: In summary, our study is the first to show activation of the inflammasome in burned humans, and our results provide impetus for further investigation of the role of the inflammasome in burn-induced hypermetabolism and, potentially, developing novel therapies targeting this protein complex for the treatment of stress-induced diabetes.

Altered neuroendocrine status at the onset of CNS lupus-like disease.

Neuropsychiatric (NP) manifestations and brain atrophy are common, etiologically unexplained complications of the systemic autoimmune disease lupus erythematosus (SLE). Similar to patients with NP SLE, behavioral deficits and neurodegeneration occur in aged, lupus-prone MRL/lpr mice. In order to gain a better understanding of the time course and nature of CNS involvement, we compare the neuro-immuno-endocrine profiles of two lupus-prone MRL/lpr stocks, which differ in disease onset and severity. Mice from stock 485 (characterized by early lupus-like manifestations) display blunted responsiveness to palatable solutions and impaired nocturnal activity as early as 7 weeks of age. They also have increased IgG in cerebrospinal fluid (CSF) before high serum autoantibody levels and splenomegaly are detected. Moreover, when compared to age-matched 6825 controls, 485 mice exhibit elevated serum corticosterone, enlarged left adrenal gland, and enhanced haematoxylin/eosin staining in the hypothalamic paraventricular nucleus. Swimming speed and novel object exploration become impaired only when more severe peripheral manifestations are documented in 17 week-old 485 mice. The obtained results suggest that performance deficits during the prodromal phase of NP SLE-like disease are associated with autoantibodies in CSF and asymmetric activation of the hypothalamus-pituitary-adrenal axis. Subsequent deterioration in behavioral performance evolves alongside systemic autoimmunity and inflammation. Although a leaky blood-CSF barrier is a possible explanation, one may hypothesize that, similar to neonatal lupus, maternal antibodies to brain antigens cross blood-placental barrier during embryogenesis and induce early endocrine and behavioral deficits in offspring.

Intrathecal antibodies and brain damage in autoimmune MRL mice.

Neuropsychiatric (NP) manifestations and brain pathology are poorly understood and potentially fatal concomitants of systemic lupus erythematosus (SLE). For many years, autoantibodies to brain tissue (i.e., brain-reactive antibodies, BRA) were proposed as a key factor in pathogenesis of CNS manifestations. Recent evidence suggests that intrathecal BRA, rather than serum autoantibodies, are a better predictor of disturbed brain morphology and function. We presently test this hypothesis by examining the relationship among BRA in cerebrospinal fluid (CSF), behavioral deficits, and brain pathology in a well-established animal model of CNS lupus. We showed earlier that significant diversity in disease manifestations within genetically homogenous MRL-lpr mice allows for constructive and informative correlational analysis. Therefore, levels of CSF antibodies were presently correlated with behavioral, neuropathological and immune measures in a cohort of diseased MRL-lpr males (N=40). ELISA, Western Blotting, standardized behavioral battery, digital planimetry, HE staining, and immunohistochemistry were employed in overall data collection. The IgG antibodies from CSF were binding to different regions of brain parenchyma, with dentate gyrus, amygdale, and subventricular zones showing enhanced immunoreactivity. High levels of CSF antibodies correlated with increased immobility in the forced-swim test and density of HE(+) cells in the paraventricular nucleus. Peripheral measures of autoimmunity were associated with other deficits in behavior and neuropathology. This correlation pattern suggests that etiology of brain damage in lupus-prone mice is multifactorial. Intrathecal BRA may be important in altering motivated responses and activity of major neuroendocrine axes at the onset of SLE-like disease.

Disturbed distribution of proliferative brain cells during lupus-like disease.

Brain atrophy and neuronal degeneration of unknown etiology are frequent and severe concomitants of the systemic autoimmune disease lupus erythematosus (SLE). Using the murine MRL/lpr model, we examined populations of proliferative brain cells during the development of SLE-like disease and brain atrophy. The disease onset was associated with reduced expression of Ki67 and BrdU proliferation markers in the dorsal part of the rostral migratory stream, enhanced Fluoro Jade C staining in the subgranular zone of the dentate gyrus, and paradoxical increase in density of Ki67(+)/BrdU(-) cells in the paraventricular nucleus. Protuberances containing clusters of BrdU(+) cells were frequent along the lateral ventricles and in some cases were bridging ventricular walls. Cells infiltrating the choroid plexus were Ki67(+)/BrdU(+), suggesting proliferative leukocytosis in this cerebrospinal fluid-producing organ. The above results further support the hypothesis that systemic autoimmune disease induces complex CNS pathology, including impaired neurogenesis in the hippocampus. Moreover, changes in the paraventricular nucleus implicate a metabolic dysfunction in the hypothalamus-pituitary-adrenal axis, which may account for altered hormonal status and psychiatric manifestations in SLE.

NLRP3 Inflammasome Modulates Post-Burn Lipolysis and Hepatic Fat Infiltration via Fatty Acid Synthase.

Burns result in generalized catabolism, lipolysis, and hyperinflammation. NLRP3 inflammasome, a mediator of hyperinflammation, is upregulated in burn patients' adipose tissue within 7 days post-burn. However, its role during the acute phase is unknown. Here, wild-type (WT) and NLRP3 knockout (NLRP3-/-) mice were exposed to 25% TBSA scald burn. Flow cytometric analysis demonstrated greater liver macrophage infiltration in NLRP3-/- yet decreased protein expression of NLRP3 components, ER stress, and apoptosis. NLRP3-/- had increased circulating free fatty acids (FFA), fatty deposition and liver weight 1 hour post-burn. Alterations in adipose fatty acid synthase (Fasn) expression affects FFA levels post-burn; WT have an early peak in Fasn gene and protein expression that is lost in NLRP3-/-, resulting in increased lipolysis and hepatic fatty deposition. In summary, our findings reveal that NLRP3 inflammasome activation is a double-edged sword. While prolonged inflammation and long-term effects of macrophage activation are associated with poor outcomes, acute inflammation may be beneficial. These results highlight the important metabolic role that NLRP3 inflammasome plays in the acute phase, ultimately affecting survival post-burn.

Status and Challenges of Predicting and Diagnosing Sepsis in Burn Patients.

Burns are a common form of trauma that account for more than 300,000 deaths each year worldwide. Survival rates have improved over the past decades because of improvements in nutritional and fluid support, burn wound care, and infection control practices. Death, however, remains unacceptably high. The primary cause of death has changed over the last decades from anoxic causes to now predominantly infections and sepsis. Sepsis and septic complications are not only major contributors to poor outcomes, but they further result in longer hospital stay and higher healthcare costs. Despite the importance of infections and sepsis, the diagnosis and prediction remain a major challenge. To date, no clear diagnostic criteria or predictive formula exist that can predict reliably the occurrence of sepsis and infections. This review will highlight and discuss current definitions and criteria for diagnosis as well as predictive biomarkers of sepsis in patients with burns. It will also present the diagnostic tools employed, such as procalcitonin, C-reactive protein, and cytokines. We will discuss the benefits and shortcomings of different treatment modalities in the context of sepsis prevention. Last, we identify new therapeutic strategies for sepsis prediction and present future considerations to prevent sepsis in patients with burns. Minimizing and preventing septic complications through early detection would significantly benefit patients and necessitate continued research to unravel new biomarkers and mechanisms. Subsequent studies need to take a fresh perspective and consider the implementation of patient-centered therapeutic strategies.

Septic predictor index: A novel platform to identify thermally injured patients susceptible to sepsis.

BACKGROUND: During the past decades' sepsis has become the major cause of death in severely burned patients. Despite the importance of burn sepsis, its diagnosis, let alone its prediction, is difficult if not impossible. Recently, we have demonstrated burn patients have increased NLRP3 inflammasome activation in white adipose tissue. We aimed to delineate a unique immune profile that can be used to identify septic outcomes in severely burned patients. METHODS: Adult burn patients (n = 37) admitted to our burn center between June 2013-2015 were enrolled in this study. White adipose tissue from the site of injury and plasma were collected from severely burned patients (>20% total body surface area) within 96 hours after thermal injury, indiscriminate of sex or age. RESULTS: We found that patients exhibiting aberrantly high levels of proinflammatory interleukin-1ß and decreased macrophages at the site of injury are highly susceptible to development of sepsis. Septic patients also had increased anti-inflammatory (interleukin-10, interleukin-1RA) cytokines in plasma. The Septic Predictor Index was generated as a quotient for the site of injury macrophage proportion and interleukin-1ß production. All patients who eventually develop sepsis had septic predictor index values >0.5. Septic patients with Septic Predictor Index values >1 all had sepsis onset within 12 days post-injury, whereas patients with Septic Predictor Index values between 0.5-1 all had later onset (>12 days). CONCLUSION: The Septic Predictor Index can determine sepsis onset accurately in thermally injured patients a priori and further enables surgeons to develop clinical studies and focused therapies specifically designed for septic cohorts.

Antioxidant and Trace Element Supplementation Reduce the Inflammatory Response in Critically Ill Burn Patients.

Oxidative stress after burn injury induces inflammatory and hypermetabolic responses associated with adverse outcomes. We propose that antioxidant and trace element supplementation may reduce oxidative stress and subsequently alleviate inflammation and hypermetabolism, thus improving clinical outcomes. We conducted a cohort study of adult patients with an acute burn injury admitted to our provincial burn center. Patients in the antioxidant group received an intravenous infusion of multivitamins and trace elements for the first 14 days after admission. The inflammatory profile was assessed at early time points, < 14 days postburn, and later time points, ≥ 15 days postburn, and included interleukin (IL)-1ß, interferon-γ, IL-1 receptor antagonist, IL-6, granulocyte-macrophage colony-stimulating factor, and FMS-like tyrosine kinase 3 ligand. Hypermetabolism was assessed by resting energy expenditure. Clinical outcomes included mortality, morbidities, hospital length of stay, and infections including days to the last positive culture after injury. We studied 172 patients, mean age 49 ± 17 years and 33 ± 13% TBSA burned, with 91 controls and 81 patients in the antioxidant group. Patients in the antioxidant group had significantly lower levels of inflammatory markers at both early and late time points, P < .05. Antioxidant treatment was associated with decreased measure of hypermetabolism, P < .05. Morbidity and mortality were not significantly different between groups. Length of hospital stay was significantly shorter in the antioxidant group when adjusted for patient demographics and injury characteristics (risk ratio (RR), 0.78; 95% confidence interval (CI), 0.66-0.92). In the antioxidant group, while infections were not different, the last positive culture post-injury was documented at median 19 days (Interquartile range (IQR), 11-43 days) compared with controls at 35 days (IQR, 15-59 days), P = .012. Patients receiving antioxidant and trace element supplementation had reduced markers of burn stress-induced inflammation; they were also associated with a decreased hypermetabolic response, shorter length of stay, and improved bacterial clearance.

Modeling Acute ER Stress in Vivo and in Vitro.

The endoplasmic reticulum (ER) is a critical organelle that synthesizes secretory proteins and serves as the main calcium storage site of the cell. The accumulation of unfolded proteins at the ER results in ER stress. Although the association between ER stress and the pathogenesis of many metabolic conditions have been well characterized using both in vivo and in vitro models, no standardized model concerning ER stress exists. Here, we report a standardized model of ER stress using two well-characterized ER stress-inducing agents, thapsigargin and tunicamycin. Our aim in this current study was 2-fold: to characterize and establish which agent is optimal for in vitro use to model acute ER stress and to evaluate which agent is optimal for in vivo use. To study the first aim we used two well-established metabolic cell lines; human hepatocellular carcinoma (HepG2s) and differentiated mouse adipocytes (3T3-L1). In the second aim we utilized C57BL/6J mice that were randomized into three treatment groups of sham, thapsigargin, and tunicamycin. Our in vitro results showed that tunicamycin worked as a rapid and efficacious inducer of ER stress in adipocytes consistently, whereas thapsigargin and tunicamycin were equally effective in inducing ER stress in hepatocytes. In regards to our in vivo results, we saw that tunicamycin was superior in not only inducing ER stress but also recapturing the metabolic alterations associated with ER stress. Thus, our findings will help guide and inform researchers as to which ER stress agent is appropriate with regards to their model.

IL-6 Signal From the Bone Marrow is Required for the Browning of White Adipose Tissue Post Burn Injury.

The hypermetabolic stress response after burn contributes to multi-organ failure, sepsis, morbidity, and mortality. The cytokine interleukin 6 (IL-6) has been hypothesized to mediate not only white adipose tissue (WAT) browning in burns, but also other hypermetabolic conditions. In addition to its inflammatory effects, IL-6 also acts as a metabolic mediator that affects metabolic tissues. Therefore, we sought to uncover the origin of circulating IL-6 post burn injury that regulates WAT browning. WAT and sera samples were collected from both adult burn patients admitted to the Ross Tilley Burn Centre at Sunnybrook Hospital and mice subjected to a burn injury. Collected tissues were analyzed for browning markers and metabolic state via histology, gene expression, and resting energy expenditure. Increased WAT browning was observed in burn patients as well as mice subjected to burn injury. Circulating IL-6 levels were significantly elevated post burn injury in mice (<0.05) and in burn patients (<0.05), the latter of which was positively correlated with elevated REE. Genetic loss of whole body IL-6 in mice prevented burn-induced WAT browning. Transplanting IL-6 knockout (KO) mice with bone marrow (BM) from wild-type (WT) mice, recovered the browning phenotype in these mice, as evaluated by increased uncoupling protein 1 (UCP1) expression (<0.05). Conversely, transplanting irradiated WT mice with BM from IL-6 KO mice impaired burn induced browning with no significant expression of UCP1. Together, our findings implicate BM derived IL-6 as the source controlling browning of WAT post burn injury. Thus, targeting IL-6 is a promising target for hypermetabolism in burns.