Lactate shuttling drives the browning of white adipose tissue after burn.

High levels of plasma lactate are associated with increased mortality in critically injured patients, including those with severe burns. Although lactate has long been considered a waste product of glycolysis, it was recently revealed that it acts as a potent inducer of white adipose tissue (WAT) browning, a response implicated in mediating postburn cachexia, hepatic steatosis, and sustained hypermetabolism. Despite the clinical presentation of hyperlactatemia and browning in burns, whether these two pathological responses are linked is currently unknown. Here, we report that elevated lactate plays a causal signaling role in mediating adverse outcomes after burn trauma by directly promoting WAT browning. Using WAT obtained from human burn patients and mouse models of thermal injury, we show that the induction of postburn browning is positively correlated with a shift toward lactate import and metabolism. Furthermore, daily administration of l-lactate is sufficient to augment burn-induced mortality and weight loss in vivo. At the organ level, increased lactate transport amplified the thermogenic activation of WAT and its associated wasting, thereby driving postburn hepatic lipotoxicity and dysfunction. Mechanistically, the thermogenic effects of lactate appeared to result from increased import through MCT transporters, which in turn increased intracellular redox pressure, [NADH/NAD+], and expression of the batokine, FGF21. In fact, pharmacological inhibition of MCT-mediated lactate uptake attenuated browning and improved hepatic function in mice after injury. Collectively, our findings identify a signaling role for lactate that impacts multiple aspects of postburn hypermetabolism, necessitating further investigation of this multifaceted metabolite in trauma and critical illness.NEW & NOTEWORTHY To our knowledge, this study was the first to investigate the role of lactate signaling in mediating white adipose tissue browning after burn trauma. We show that the induction of browning in both human burn patients and mice is positively correlated with a shift toward lactate import and metabolism. Daily l-lactate administration augments burn-induced mortality, browning, and hepatic lipotoxicity in vivo, whereas pharmacologically targeting lactate transport alleviates burn-induced browning and improves liver dysfunction after injury.

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.

Single-nuclei RNA Profiling Reveals Disruption of Adipokine and Inflammatory Signaling in Adipose Tissue of Burn Patients.

OBJECTIVE: We conducted a large-scale investigation of the systemic and adipose tissue-specific alterations in a clinical population of burn patients to identify factors that may influence hypermetabolism. BACKGROUND: Previous research has identified chronic disturbances in adipose tissue inflammation, lipolysis, and browning, which may drive the perpetuation of hypermetabolism following the severe adrenergic stress of a burn injury. Given that adipose tissue is thought to be a central node in the regulation of systemic metabolism, we believe that systematically delineating the pathologic role of adipose tissue postburn, will lead to the identification of novel interventions to mitigate morbidity and mortality from severe burns. METHODS: This was a single-institution cohort study, which obtained plasma and subcutaneous adipose tissue samples from severely burn adult patients over various time points during acute hospitalization. Whole-body clinical, metabolic, and inflammatory mediators were assessed in plasma, while genetic analyses through RT-qPCR and single-nuclei RNA sequencing were conducted in adipose tissue. RESULTS: Systemic inflammation and adrenergic stress increase IL-6 signaling, lipolysis, browning, and adipokine dysfunction in the adipose tissue of adult burn patients, which may further propagate the long-term hypermetabolic response. Moreover, using single-nuclei RNA sequencing, we provide the first comprehensive characterization of alterations in the adipose tissue microenvironment occurring at acute and chronic stages postburn. CONCLUSION: We provide novel insight toward the effect of burns on adipokine release, inflammatory signaling pathways, and adipose heterogeneity over the trajectory of acute and chronic stages.

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.

Interleukin-6 blockade, a potential adjunct therapy for post-burn hypermetabolism.

Severe burns remain a leading cause of death and disability worldwide. Despite advances in patient care, the excessive and uncontrolled hypermetabolic stress response induced by this trauma inevitably affects every organ system causing substantial morbidity and mortality. Recent evidence suggests interleukin-6 (IL-6) is a major culprit underlying post-burn hypermetabolism. Indeed, genetic deletion of IL-6 alleviates various complications associated with poor clinical outcomes including the adverse remodeling of adipose tissue, cachexia and hepatic steatosis. Thus, pharmacological blockade of IL-6 may be a more favorable treatment option to fully restore metabolic function after injury. To test this, we investigated the safety and effectiveness of blocking IL-6 for post-burn hypermetabolism using a validated anti-IL-6 monoclonal antibody (mAb) in our experimental murine model. Here, we show daily anti-IL-6 mAb administration protects against burn-induced weight loss (P < .0001) without any adverse effect on mortality. At the organ level, post-burn treatment with the IL-6 blocker suppressed the thermogenic activation of adipose tissue (P < .01) and its associated wasting (P < .05). The reduction of browning-induced lipolysis (P < .0001) indirectly decreased hepatic lipotoxicity (P < .01) which improved liver dysfunction (P < .05). Importantly, the beneficial effects of this anti-IL-6 agent extended to the skin, reflected by the decrease in excessive collagen deposition (P < .001) and genes involved in pathologic fibrosis and scarring (P < .05). Together, our results indicate that post-burn IL-6 blockade leads to significant improvements in systemic hypermetabolism by inhibiting pathological alterations in key immunometabolic organs. These findings support the therapeutic potential of anti-IL-6 interventions to improve care, quality of life, and survival in burned patients.

Activation of ER stress signalling increases mortality after a major trauma.

The endoplasmic reticulum (ER) adapts to stress by activating a signalling cascade known as the ER stress response. While ER stress signalling is a central component of the cellular defence against environmental insult, persistent activation is thought to contribute to the progression of various metabolic complications via loss of protein function and cell death. Despite its importance however, whether and how ER stress impacts morbidity and mortality in conditions of hypermetabolism remain unclear. In this study, we discovered that chronic ER stress response plays a role in mediating adverse outcomes that occur after major trauma. Using a murine model of thermal injury, we show that induction of ER stress with Tunicamycin not only increased mortality but also resulted in hepatic damage and hepatic steatosis. Importantly, post-burn treatment with chaperone ER stress inhibitors attenuated hepatic ER stress and improved organ function following injury. Our study identifies ER stress as a potential hub of the signalling network affecting multiple aspects of metabolism after major trauma and as a novel potential molecular target to improve the clinical outcomes of severely burned patients.

NLRP3 inflammasome mediates white adipose tissue browning after burn.

A hallmark after burn is the stress and inflammatory-induced hypermetabolic response. Recently, we and others found that browning of white adipose tissue (WAT) is a critical component of this complex detrimental response. Although browning and inflammation have been independently delineated to occur after injury, their interaction is currently not well defined. One of the master regulators of inflammation and adipose tissue remodeling after burns is nucleotide-binding and oligomerization domain, leucine rich repeat and pyrin domain containing 3 (NLRP3) inflammasome. The aim of this this study was to determine whether NLRP3 modulates and activates WAT browning after burn. To obtain molecular and mechanistic insights, we used an NLRP3 knockout (NLRP3-/-) murine burn model. We demonstrated that genetic deletion of NLRP3 promoted persistent and augmented browning in adipocytes, evidenced by increased gene expression of peroxisome proliferator-activated receptor γ and CIDEA at 3 days (5.74 vs. 0.29, P < 0.05; 26.0 vs. 0.71, P < 0.05) and uncoupling protein 1 (UCP1) and PGC1α at 7 days (7,406 vs. 3,894, P < 0.05; 20.6 vs. 2.52, P < 0.01) and enhanced UCP1 staining and multilocularity. Additionally, the main regulator of postburn WAT browning, IL-6, was elevated in the plasma acutely after burn in NLRP3-/- compared with wild-type counterparts (478.9 vs. 67.1 pg/mL, P < 0.05 at 3 days). These results suggest that NLRP3 has antibrowning effects and that blocking NLRP3 increases thermogenesis and augments browning via increased levels of IL-6. Our findings provide insights into targeting innate inflammatory systems for regulation of adaptive thermogenesis, a critical response after burns and other hypermetabolic conditions.

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.

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.

Hepatic mitochondrial bioenergetics in aged C57BL/6 mice exhibit delayed recovery from severe burn injury.

Severe burn injuries initiate a cascade of downstream events, culminating in multiple organ dysfunction, sepsis, and even death. The elderly are in particular vulnerable to such outcomes, due primarily to a scarcity of knowledge on trauma progression at the biomolecular level in this population. Mitochondria, the cellular powerhouses, have been increasingly scrutinized recently for their contribution to trauma outcomes. We hypothesized that elderly have a worse outcome compared to adult patients due to failed recovery of hepatic mitochondria. Using a murine model of burn injury, Seahorse respirometry and functional proteomic assays, we demonstrate the impact of thermal trauma on hepatic mitochondrial respiration in adult and aged mice. While the mitochondria in adults rebound from the initial insult within 7days of the injury, the older animals display delayed recovery of mitochondrial bioenergetics accompanied by uncoupling and an oxidative environment. This is associated with a state of increased protein oxidation and nitrosylation, along with increases in circulating mtDNA, a known damage-associated molecular pattern. These findings suggest that hepatic mitochondria fail to normalize after trauma in aged mice and we suggest that this cellular failure is associated with organ damage and subsequently increased morbidity and mortality in elderly burn patients.

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.

Endoplasmic reticulum stress in adipose tissue augments lipolysis.

The endoplasmic reticulum (ER) is an organelle important for protein synthesis and folding, lipid synthesis and Ca(2+) homoeostasis. Consequently, ER stress or dysfunction affects numerous cellular processes and has been implicated as a contributing factor in several pathophysiological conditions. Tunicamycin induces ER stress in various cell types in vitro as well as in vivo. In mice, a hallmark of tunicamycin administration is the development of fatty livers within 24-48 hrs accompanied by hepatic ER stress. We hypothesized that tunicamycin would induce ER stress in adipose tissue that would lead to increased lipolysis and subsequently to fatty infiltration of the liver and hepatomegaly. Our results show that intraperitoneal administration of tunicamycin rapidly induced an ER stress response in adipose tissue that correlated with increased circulating free fatty acids (FFAs) and glycerol along with decreased adipose tissue mass and lipid droplet size. Furthermore, we found that in addition to fatty infiltration of the liver as well as hepatomegaly, lipid accumulation was also present in the heart, skeletal muscle and kidney. To corroborate our findings to a clinical setting, we examined adipose tissue from burned patients where increases in lipolysis and the development of fatty livers have been well documented. We found that burned patients displayed significant ER stress within adipose tissue and that ER stress augments lipolysis in cultured human adipocytes. Our results indicate a possible role for ER stress induced lipolysis in adipose tissue as an underlying mechanism contributing to increases in circulating FFAs and fatty infiltration into other organs.

CLASSIC IL-6 SIGNALING IS ASSOCIATED WITH POOR OUTCOMES IN BURN PATIENTS.

ABSTRACT: Background: Interleukin (IL)-6 is a multifunctional cytokine with both a proinflammatory and anti-inflammatory role. In many studies, IL-6 increases rapidly after burn injury and is associated with poor outcomes. However, there are two aspects to IL-6; it can signal via its soluble IL-6 receptor (sIL-6R), which is referred to as trans-signaling and is regarded as the proinflammatory pathway. The role of sIL-6R postburn injury has yet to be explored in its entirety. We hypothesized that patients with a lower ratio of IL-6 to sIL-6R would have worse outcomes. Methods: Patients admitted to our burn center within 7 days of injury were included in this study. Patients were divided into two groups based on IL-6 and sIL-6R levels measured within the first 7 days postburn injury. Patients were in the high ratio group if their IL-6/sIL-6R ratio was ≥0.185. Clinical outcomes included organ biomarkers, morbidities, and hospital length of stay. Groups were compared using Student's t test, Mann-Whitney U , and Fisher's exact test as appropriate; a P value of <0.05 was considered statistically significant. Results: We studied 86 patients with a median age of 50 years (36-66 years) and a median total body surface area burn of 18% (10-31). There were 40 patients categorized with a low IL-6/sIL-6R ratio and 46 patients with a high IL-6/sIL-6R ratio. Patients in the high IL-6/sIL-6R ratio group had a significantly greater total body surface area burn ( P < 0.001) and a significantly greater proportion of patients with inhalation injury ( P = 0.001). Levels of IL-6 were significantly higher in patients with a high IL-6/sIL-6R ratio ( P < 0.0001). However, levels of sIL-6R were not significantly different among the low and high groups ( P = 0.965). Mortality was significantly greater in the high IL-6/sIL-6R ratio group (3% vs. 26%; P = 0.002). Conclusions: Interestingly, patients with a higher ratio of IL-6/sIL-6R had significantly greater mortality. Using sIL-6R as a marker for the proinflammatory immune response, we expected patients with a lower IL-6/sIL-6R ratio to have poor outcomes, typically associated with a hyperinflammatory or exaggerated immune response. However, the absolute value of sIL-6R did not differ. This suggests that classical signaling of IL-6 via its membrane-bound receptor, with an anti-inflammatory function, is important.

Aging Impairs the Cellular Interplay between Myeloid Cells and Mesenchymal Cells during Skin Healing in Mice.

Impaired wound healing is a major issue in the elderly population and is associated with substantial health and economic burden, which is exponentially increasing with the growing aging population. While the underlying pathobiology of disturbed skin healing by aging is linked to several genetic and epigenetic factors, little is known about the cell-cell interaction during the wound healing process in aged individuals, particularly the mesenchymal stem cell (MSCs)-macrophages axis. In this study, by using a thermal injury animal model in which we compared the wound healing process of adult and young mice, we found that the insufficient pool of MSCs in adult animals are deficient in migrating to the wound bed and instead are restricted to the wound edge. We identified a deficiency of a CD90-positive MSC subpopulation in the wounds of adult animals, which is positively correlated with the number of F4/80+ macrophages. In vitro, we found that CD90+ cells preferentially adhere to the myeloid cells forming doublet cells. Thus, our findings highlight that in adult mice subjected to a thermal injury, impaired wound healing is likely mediated by a disturbed cellular interplay between myeloid cells and mesenchymal cells.

Thermal Stress Induces Long-Term Remodeling of Adipose Tissue and Is Associated with Systemic Dysfunction.

ABSTRACT: Severe burns are characterized by the magnitude and duration of the hypermetabolic response thereafter, and demarcated by the loss of lean body mass and catabolism of fat stores. The aim of the present study was to delineate the temporal and location-specific physiological changes to adipose depots and downstream consequences post-burn in a murine model of thermal injury. C57BL/6 mice were subjected to a 30% total body surface area burn and body mass, food intake, and tissue mass were monitored for various time points up until 60 days postinjury. Mitochondrial respirometry was performed using a Seahorse XF96 analyzer. Lipolytic markers and browning markers were analyzed via Western blotting and histology. A severe burn results in a futile cycle of lipolysis and white adipose tissue (WAT) browning, the sequelae of which include fat catabolism, hepatomegaly, and loss of body mass despite increased food intake. A dynamic remodeling of epididymal WAT was observed with acute and chronic increases in lipolysis. Moreover, we demonstrate that pathological browning of inguinal WAT persists up to 60 days post-burn, highlighting the magnitude of the ß-adrenergic response to thermal injury. Our data suggests that adipose depots have a heterogeneous response to burns and that therapeutic interventions targeting these physiological changes can improve outcomes. These data may also have implications for treating catabolic conditions such as cancer cachexia as well as developing treatments for obesity and type II diabetes.

Adipose browning response to burn trauma is impaired with aging.

BACKGROUNDThe incidence of burn injuries in older patients is dramatically increasing as the population of older people grows. Despite the increased demand for elderly burn care, the mechanisms that mediate increased morbidity and mortality in older trauma patients are unknown. We recently showed that a burn injury invokes white adipose tissue browning that leads to a substantially increased hypermetabolic response associated with poor outcomes. Therefore, the aim of this study was to determine the effect of age on the metabolic adipose response of browning after a burn injury.METHODOne hundred and seventy patients with burn injury admitted to the Ross Tilley Burn Centre were prospectively enrolled and grouped by age as older (≥50 years) and young (≤35 years). Adipose tissue and sera were collected and analyzed for browning markers and metabolic state via histology, gene expression, and resting energy expenditure assays.RESULTSWe found that older patients with burn injury lacked the adipose browning response, as they showed significant reductions in uncoupling protein 1 (UCP1) expression. This failure of the browning response was associated with reduced whole-body metabolism and decreased survival in older patients with burn injury. Mechanistically, we found that the adipose of both aged patients after burn trauma and aged mice after a burn showed impairments in macrophage infiltration and IL-6, key immunological regulators of the browning process after a severe trauma.CONCLUSIONTargeting pathways that activate the browning response represents a potential therapeutic approach to improve outcomes after burn trauma for elderly patients.FUNDINGNIH (R01-GM087285-01), Canadian Institutes of Health Research (grant no. 123336), and Canada Foundation for Innovation Leaders Opportunity Fund (no. 25407).

NLRP3 inflammasome activity is required for wound healing after burns.

Survival of burn patients is contingent on effective wound healing, a complex process that requires coordinated responses of myeloid cells and inflammatory pathways. NLRP3, which serves as a platform for secretion of proinflammatory cytokines, is implicated as a central regulator of wound healing. However, its role during the acute dermal and epidermal regeneration in the context of burns is unknown. Wild-type (WT) and NLRP3-/- mice were exposed to a 30% TBSA scald burn. Gene expression was conducted via real-time polymerase chain reaction. Trichrome staining was used to assess collagen deposition and granulation tissue formation. F4/80 immunostaining compared macrophage infiltration. Flow cytometric analysis was used to characterize skin macrophage distribution and profile. NLRP3, IL1ß and IL18 expression was upregulated in skin after burn, and these changes were nonexistent in NLRP3-/-. NLRP3-/- had decreased expression of proinflammatory cytokines, chemokines, inflammatory markers, and growth factors at 3 days (P < 0.05). NLRP3-/- burn skin demonstrated significantly less macrophage infiltration and higher expression of anti-inflammatory markers Arg1 and Fizz1 (P < 0.05) compared to WT. Trichrome staining showed decreased collagen deposition compared to WT. We show that NLRP3 is protective in burn wound healing, primarily through production of inflammatory mediators, macrophage recruitment, and polarization to a proinflammatory phenotype. Our findings highlight a central role of NLRP3 in wound healing through regulation of inflammation and macrophage polarization after burns.

Regulation of glycolysis and the Warburg effect in wound healing.

One of the most significant adverse postburn responses is abnormal scar formation, such as keloids. Despite its prolificacy, the underlying pathophysiology of keloid development is unknown. We recently demonstrated that NLRP3 inflammasome, the master regulator of inflammatory and metabolic responses (e.g., aerobic glycolysis), is essential for physiological wound healing. Therefore, burn patients who develop keloids may exhibit altered immunometabolic responses at the site of injury, which interferes with normal healing and portends keloid development. Here, we confirmed keloid NLRP3 activation (cleaved caspase-1 [P < 0.05], IL-1ß [P < 0.05], IL-18 [P < 0.01]) and upregulation in Glut1 (P < 0.001) and glycolytic enzymes. Burn skin similarly displayed enhanced glycolysis and Glut1 expression (P < 0.01). However, Glut1 was significantly higher in keloid compared with nonkeloid burn patients (>2 SD above mean). Targeting aberrant glucose metabolism with shikonin, a pyruvate kinase M2 inhibitor, dampened NLRP3-mediated inflammation (cleaved caspase-1 [P < 0.05], IL-1ß [P < 0.01]) and improved healing in vivo. In summary, burn skin exhibited evidence of Warburg-like metabolism, similar to keloids. Targeting this altered metabolism could change the trajectory toward normal scarring, indicating the clinical possibility of shikonin for abnormal scar prevention.

Catecholamines Induce Endoplasmic Reticulum Stress Via Both Alpha and Beta Receptors.

Severely burned patients suffer from a hypermetabolic syndrome that can last for years after the injury has resolved. The underlying cause of these metabolic alterations most likely involves the persistent elevated catecholamine levels that follow the surge induced by thermal injury. At the cellular level, endoplasmic reticulum (ER) stress in metabolic tissues is a hallmark observed in patients following burn injury and is associated with several detrimental effects. Therefore, ER stress could be the underlying cellular mechanism of persistent hypermetabolism in burned patients. Here, we show that catecholamines induce ER stress and that adreno-receptor blockers reduce stress responses in the HepG2 hepatocyte cell line. Our results also indicate that norepinephrine (NE) significantly induces ER stress in HepG2 cells and 3T3L1 mouse adipocytes. Furthermore, we demonstrate that the alpha-1 blocker, prazosin, and beta blocker, propranolol, block ER stress induced by NE. We also show that the effects of catecholamines in inducing ER stress are cell type-specific, as NE treatment failed to evoke ER stress in human fibroblasts. Thus, these findings reveal the mechanisms used by catecholamines to alter metabolism and suggest inhibition of the receptors utilized by these agents should be further explored as a potential target for the treatment of ER stress-mediated disease.