Mile Stanojcic

Burn Induces Browning of the Subcutaneous White Adipose Tissue in Mice and Humans

SUMMARY Burn is accompanied by long-lasting immunometabolic alterations referred to as hypermetabolism that are characterized by a considerable increase in resting energy expenditure and substantial whole-body catabolism. In burned patients, the length and magnitude of the hypermetabolic state is the highest of all patients and associated with profoundly increased morbidity and mortality. Unfortunately, the mechanisms involved in hypermetabolism are essentially unknown. We hypothesized that the adipose tissue plays a central role for the induction and persistence of hypermetabolism post-burn injury. Here, we show that burn induces a switch in the phenotype of the subcutaneous fat from white to beige, with associated characteristics such as increased mitochondrial mass and UCP1 expression. Our results further demonstrate the significant role of catecholamines and interleukin-6 in this process. We conclude that subcutaneous fat remodeling and browning represent an underlying mechanism that explains the elevated energy expenditure in burn-induced hypermetabolism.

Pathophysiologic response to burns in the elderly

Over the last decades advancements have improved survival and outcomes of severely burned patients except one population, elderly. The Lethal Dose 50 (LD50) burn size in elderly has remained the same over the past three decades, and so has morbidity and mortality, despite the increased demand for elderly burn care. The objective of this study is to gain insights on why elderly burn patients have had such a poor outcome when compared to adult burn patients. The significance of this project is that to this date, burn care providers recognize the extreme poor outcome of elderly, but the reason remains unclear. In this prospective translational trial, we have determined clinical, metabolic, inflammatory, immune, and skin healing aspects. We found that elderly have a profound increased mortality, more premorbid conditions, and stay at the hospital for longer, p < 0.05. Interestingly, we could not find a higher incidence of infection or sepsis in elderly, p > 0.05, but a significant increased incidence of multi organ failure, p < 0.05. These clinical outcomes were associated with a delayed hypermetabolic response, increased hyperglycemic and hyperlipidemic responses, inversed inflammatory response, immune-compromisation and substantial delay in wound healing predominantly due to alteration in characteristics of progenitor cells, p < 0.05. In summary, elderly have substantially different responses to burns when compared to adults associated with increased morbidity and mortality. This study indicates that these responses are complex and not linear, requiring a multi-modal approach to improve the outcome of severely burned elderly.

Stress Hyperglycemia, Insulin Treatment, and Innate Immune Cells

Hyperglycemia (HG) and insulin resistance are the hallmarks of a profoundly altered metabolism in critical illness resulting from the release of cortisol, catecholamines, and cytokines, as well as glucagon and growth hormone. Recent studies have proposed a fundamental role of the immune system towards the development of insulin resistance in traumatic patients. A comprehensive review of published literatures on the effects of hyperglycemia and insulin on innate immunity in critical illness was conducted. This review explored the interaction between the innate immune system and trauma-induced hypermetabolism, while providing greater insight into unraveling the relationship between innate immune cells and hyperglycemia. Critical illness substantially disturbs glucose metabolism resulting in a state of hyperglycemia. Alterations in glucose and insulin regulation affect the immune function of cellular components comprising the innate immunity system. Innate immune system dysfunction via hyperglycemia is associated with a higher morbidity and mortality in critical illness. Along with others, we hypothesize that reduction in morbidity and mortality observed in patients receiving insulin treatment is partially due to its effect on the attenuation of the immune response. However, there still remains substantial controversy regarding moderate versus intensive insulin treatment. Future studies need to determine the integrated effects of HG and insulin on the regulation of innate immunity in order to provide more effective insulin treatment regimen for these patients.

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

ObjectivesSevere 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. DesignProspective cohort study. SettingRoss Tilley Burn Centre & Sunnybrook Research Institute. PatientsWe 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. InterventionsNone. 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-1&bgr; 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.

Norepinephrine Inhibits Macrophage Migration by Decreasing CCR2 Expression

Increased incidences of infectious and septic complications during post-burn courses represent the main contributor to burn injury mortality. Sustained increases in catecholamine levels, especially norepinephrine (NE), contribute to immune disturbances in severely burned patients. The precise mechanisms underlying NE-mediated immunoregulation are not fully understood. Here we hypothesize that persistently elevated NE levels are associated with immunodysfunctions. We examined the effects of NE on the phenotype and functions of bone marrow-derived macrophages (BMMs). Whole mouse bone marrow cells were treated in vitro with 40 ng/mL of M-CSF and with 1 x 10-6 M or 1 x 10-8 M of NE or without NE for 7 days; cells were collected and stained with antibodies for CD11b, F4/80, MHC II and the inflammatory CC chemokine receptor 2 (CCR2). We found 1 x 10-6 M of NE inhibited MHC II and CCR2 expression on CD11b+/F4/80+ BMM cells. It also inhibited BMM proliferation by inhibiting CSF-1R expression. On the contrary, 1 x 10-8 M of NE slightly increased both MHC II and CCR2 expression on CD11b+/F4/80+ BMM cells but inhibited CD11b+/F4/80+ BMM proliferation. MCP-1 based migration assay showed that the migration of 1 x 10-6 M of NE-treated BMM toward MCP-1 was significantly decreased compared to BMM without NE treatment. Both 1 x 10-8 M and 1 x 10-6 M of NE enhanced TNF-α production and phagocytosis of FITC-Dextran. Intracellular staining of transcriptional factor MafB showed that 1 x 10-6 M of NE treatment enhanced its expression, whereas 1 x 10-8 M of NE decreased expression. Stimulation with LPS in the last 24-hours of BMM culture further decreased CCR2 and MHC II expression of these BMM, suggesting the synergistic effect of LPS and NE on macrophage. Our results demonstrate that NE regulates macrophage differentiation, proliferation and function, and may play a critical role in the dysfunctional immune response post-burn.

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.

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

ABSTRACT 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.

Prolonged Endoplasmic Reticulum-Stressed Hepatocytes Drive an Alternative Macrophage Polarization.

ABSTRACT Relatively little is known about the effects of hepatocytes on hepatic macrophages, particularly under the situation of endoplasmic reticulum (ER) stress. We examined the effects of hepatocytes conditioned media (CM) from HepG2 treated with ER stress inducers, tunicamycin or thapsigargin, on the secretion of cytokines, expression of ER stress markers, and polarization of phorbol myristate acetate–activated THP-1 cells (pTHP-1). We found that CM decreased the production of the proinflammatory cytokines including tumor necrosis factor &agr;, interleukin 6 (IL-6), and IL-1&bgr; as well as other cytokines and chemokines from pTHP-1 cells. These effects are mediated by the inhibition of TLR4 expression and nuclear factor &kgr;B signaling pathway. In addition, hepatocytes CM increased the expression of binding immunoglobulin protein and the transcription factor C/EBP homologous protein (CHOP) in pTHP-1 cells. Preconditioning with ER stress inhibitor, small molecular chaperone 4-phenylbutyrate before addition of ER stressors, attenuated the ER stress in macrophages, the property of hepatocytes CM to alter tumor necrosis factor &agr; production and nuclear factor &kgr;B expression by macrophages. Remarkably, treatment of macrophage with these CM leads to an alternative activation of macrophages mediated by peroxisome proliferator-activated receptor &ggr; signaling pathway, which might be resulted from the secretion of IL-10 and IL-4 as well as releasing apoptotic bodies from hepatocytes under ER stress. Our results highlight a mechanism of ER stress transmission from hepatocytes to macrophage that drives an alternative activation of macrophages, which depends on the exposure of hepatocytes to severe and prolonged ER stress.

Induction of antigen-specific TH9 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 TH9 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 TH9 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 TH2 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.