Myosin light chain phosphorylation and pulmonary endothelial cell hyperpermeability in burns

Major cutaneous burns result in not only localized tissue damage but broad systemic inflammation causing organ system damage distal to the burn site. It is well recognized that many problems result from the release of inflammatory mediators that target vascular endothelial cells, causing organ dysfunction. The pulmonary microvessels are particularly susceptible to functional abnormalities as a direct consequence of exposure to burn-induced inflammatory mediators. Traditional therapeutic intervention is quite often ineffective in treating burn patients suffering from systemic problems. A possible explanation for this ineffectiveness may be that because so many mediators are released, supposedly activating numerous signaling cascades that interact with each other, targeting of upstream factors in these cascades on an individual basis becomes futile. Therefore, if an end-point effector responsible for endothelial dysfunction following burn injury could be identified, it may present a target for intervention. In this study, we identified phosphorylation of myosin light chain (MLC) as a required element of burn plasma-induced hyperpermeability across rat lung microvascular endothelial cell monolayers. In addition, pharmacological inhibition of myosin light chain kinase (MLCK) and Rho kinase as well as transfection of MLCK-inhibiting peptide blocked actin stress fiber formation and MLC phosphorylation in response to burn plasma. The results suggest that blocking MLC phosphorylation may provide therapeutic intervention in burn patients with the goal of alleviating systemic inflammation-induced endothelial dysfunction.     

Ghrelin stimulates food intake and growth hormone release in rats with thermal injury: synthesis of ghrelin

Ghrelin, a 28-residue octanoylated peptide recently isolated from the stomach, exhibits anti-cachectic properties through regulating food intake, energy expenditure, adiposity, growth hormone secretion and immune response. Burn injury induces persistent hypermetabolism and muscle wasting. We therefore hypothesized that ghrelin may also play a role in the pathophysiology of burn-induced cachexia. Overall ghrelin expression in the stomach over 10 days after burn was significantly decreased (p = 0.0003). Total plasma ghrelin was reduced 1 day after burn. Thus, changes in ghrelin synthesis and release may contribute to burn-induced dysfunctions. Ghrelin (30 nmol/rat, i.p.) greatly stimulated 2 h food intake in rats on five separate days after burn and in control rats. On post-burn day 15, plasma growth hormone levels were significantly lower than in controls, and this was restored to normal levels by ghrelin (10 nmol/rat, i.p.). These observations suggest that ghrelin retains its ability to favorably modulate both the peripheral anabolic and the central orexigenic signals, even after thermal injury despite ongoing changes due to prolonged and profound hypermetabolism, suggesting that long-term treatment with ghrelin may attenuate burn-induced dysfunctions.     

Psychogenic stress prior to burn injury has differential effects on bone marrow and cytokine responses

It is well known that many burn patients experience psychopathological disorders prior to burn injury. However, it is not known whether individuals that have been exposed to chronic psychological stresses will respond differently than unstressed individuals when challenged by a burn injury. In this study, we assessed whether chronic psychogenic stress prior to burn injury had any significant impact on burn injury-induced alterations in the myeloid compartment in the bone marrow and serum cytokine levels utilizing a well-controlled purely psychogenic stress model (predator exposure). Mice were individually caged and exposed to a Long Evans rat for 1 hr a day on 3 consecutive days prior to a 15% total body surface area flame burn. Four days after burn injury, bone marrow and serum were collected to assess myeloid cells and cytokine levels, respectively. Bone marrow cells were cultured in granulocyte-macrophage colony-stimulating factor (GM-CSF) to assess clonogenic ability. Flow cytometry was also used to characterize the populations of myeloid cells based on Gr-1 and CD11b staining intensity and to determine the expression of the macrophage colony-stimulating factor receptor (M-CSFR). Serum was assayed for IL-6, IL-12p70, MCP-1, and IFN-gamma by multiplexed sandwich enzyme-linked immunoabsorbent assay (ELISA). We found that predator exposure prior to burn injury ablated the burn-induced increase in myeloid colony formation and attenuated the burn-induced increases in immature monocytes and immature neutrophils in the bone marrow, as well as MCP-1 levels in the serum. Conversely, psychogenic stress exaggerated the burn-induced increase in the number of M-CSFR-positive cells. This study is the first to show the effects of a pure psychogenic stressor (predator exposure) on burn-induced alterations of the immune system. The clinical ramifications of our findings remain to be elucidated.     

TLR4 inactivation and rBPI(21) block burn-induced myocardial contractile dysfunction

Both large burns and severe gram-negative sepsis are associated with acute myocardial contractile dysfunction. Because others have reported that burn injury may be followed by transient endotoxemia, we hypothesized that bacterial endotoxin induces contractile impairment after burn trauma. We tested this hypothesis in two rodent models. In each model, postburn myocardial contractility was assessed using Langendorff preparations of excised hearts. In the first model, mice expressing either a mutant form of or no Toll-like receptor 4 (TLR4), a critical element of the mammalian endotoxin receptor, were resistant to postburn myocardial contractile dysfunction. In the second model, starting 30 min or 4 h after burn injury, rats were infused with recombinant bactericidal/permeability-increasing protein (rBPI(21)), a protein that binds and neutralizes endotoxin. Hearts from rBPI(21)-treated animals were completely protected from postburn contractile impairment. Because burn-induced contractile dysfunction can be prevented either by blocking signaling through the endotoxin receptor or by neutralizing circulating LPS, bacterial endotoxin may contribute to impaired myocardial contractility after burn injury.     

Leptin ameliorates burn-induced multiple organ damage and modulates postburn immune response in rats

The present study was designed to determine whether exogenous leptin reduces remote organ injury in the rats with thermal burn trauma. Leptin (10 microg/kg) or saline was administered intraperitoneally after burn injury, and the rats were decapitated at either 6 or 24 h. Plasma samples of 24-h burn group were assayed for the determination of monocyte and neutrophil apoptosis. Thermal injury increased tissue-associated myeloperoxidase (MPO) activity and microscopic damage scores in the lung, liver, stomach, colon and kidney of both 6- and 24-h burn groups. In the 6-h burn group, leptin reduced microscopic damage score in the liver and kidney only, while damage scores in the 24-h burn group were reduced in all the tissues except the lung. Also, in both burn groups, leptin reduced elevated MPO activity in all tissues except the lung. The percentage of mononuclear cells was significantly reduced at the 24 h of burn injury, while the granulocyte percentage was increased. Leptin treatment, however, had no significant effect on burn-induced reversal of white blood cell ratios. On the other hand, burn-induced increase in the death of mononuclear cells and granulocytes was significantly reduced in leptin-treated rats. The results of the present study suggest that leptin may provide a therapeutic benefit in diminishing burn-induced inflammation and associated multiple organ failure.     

C5a-blockade improves burn-induced cardiac dysfunction

We previously reported that generation of the anaphylatoxin C5a is linked to the development of cardiac dysfunction in sepsis due to C5a interaction with its receptor (C5aR) on cardiomyocytes. Burn injury involves inflammatory mechanisms that can lead to C5a generation as well. In this study, we investigated the effects of C5a blockade on burn-induced cardiac dysfunction. Using a standardized rat model of full thickness scald injury, left ventricular pressures were recorded in vivo followed by in vitro assessment of sarcomere contraction of single cardiomyocytes. Left ventricular pressures in vivo and cardiomyocyte sarcomere contractility in vitro were significantly reduced following burn injury. In the presence of anti-C5a Ab, these defects were greatly attenuated 1, 6, and 12 h after burn injury and completely abolished 24 h after burn. In vitro incubation of cardiomyocytes with bacterial LPS accentuated the impaired contractility, which was partially prevented in cardiomyocytes from burned rats that had received an anti-C5a Ab. Based on Western blot analyses, real-time PCR, and immunostaining of left ventricular heart tissue, there was a significant increase in cardiomyocyte expression of C5aR after burn injury. In conclusion, an in vivo blockade of C5a attenuates burn-induced cardiac dysfunction. Further deterioration of contractility due to the exposure of cardiomyocytes to LPS was partially prevented by C5a-blockade. These results suggest a linkage between C5a and burn-induced cardiac dysfunction and a possible contribution of LPS to these events.     

Extracellular Vesicle-Mediated Transfer of Genetic Information between the Hematopoietic System and the Brain in Response to Inflammation

Mechanisms behind how the immune system signals to the brain in response to systemic inflammation are not fully understood. Transgenic mice expressing Cre recombinase specifically in the hematopoietic lineage in a Cre reporter background display recombination and marker gene expression in Purkinje neurons. Here we show that reportergene expression in neurons is caused by intercellular transfer of functional Cre recombinase messenger RNA from immune cells into neurons in the absence of cell fusion. In vitro purified secreted extracellular vesicles (EVs) from blood cells contain Cre mRNA, which induces recombination in neurons when injected into the brain. Although Cre-mediated recombination events in the brain occur very rarely in healthy animals, their number increases considerably in different injury models, particularly under inflammatory conditions, and extend beyond Purkinje neurons to other neuronal populations in cortex, hippocampus, and substantia nigra. Recombined Purkinje neurons differ in their miRNA profile from their nonrecombined counterparts, indicating physiological significance. These observations reveal the existence of a previously unrecognized mechanism to communicate RNA-based signals between the hematopoietic system and various organs, including the brain, in response to inflammation.     

Role of Protein Farnesylation in Burn-Induced Metabolic Derangements and Insulin Resistance in Mouse Skeletal Muscle

Objective

Metabolic derangements, including insulin resistance and hyperlactatemia, are a major complication of major trauma (e.g., burn injury) and affect the prognosis of burn patients. Protein farnesylation, a posttranslational lipid modification of cysteine residues, has been emerging as a potential component of inflammatory response in sepsis. However, farnesylation has not yet been studied in major trauma. To study a role of farnesylation in burn-induced metabolic aberration, we examined the effects of farnesyltransferase (FTase) inhibitor, FTI-277, on burn-induced insulin resistance and metabolic alterations in mouse skeletal muscle.

Methods

A full thickness burn (30% total body surface area) was produced under anesthesia in male C57BL/6 mice at 8 weeks of age. After the mice were treated with FTI-277 (5 mg/kg/day, IP) or vehicle for 3 days, muscle insulin signaling, metabolic alterations and inflammatory gene expression were evaluated.

Results

Burn increased FTase expression and farnesylated proteins in mouse muscle compared with sham-burn at 3 days after burn. Simultaneously, insulin-stimulated phosphorylation of insulin receptor (IR), insulin receptor substrate (IRS)-1, Akt and GSK-3β was decreased. Protein expression of PTP-1B (a negative regulator of IR-IRS-1 signaling), PTEN (a negative regulator of Akt-mediated signaling), protein degradation and lactate release by muscle, and plasma lactate levels were increased by burn. Burn-induced impaired insulin signaling and metabolic dysfunction were associated with increased inflammatory gene expression. These burn-induced alterations were reversed or ameliorated by FTI-277.

Conclusions

Our data demonstrate that burn increased FTase expression and protein farnesylation along with insulin resistance, metabolic alterations and inflammatory response in mouse skeletal muscle, all of which were prevented by FTI-277 treatment. These results indicate that increased protein farnesylation plays a pivotal role in burn-induced metabolic dysfunction and inflammatory response. Our study identifies FTase as a novel potential molecular target to reverse or ameliorate metabolic derangements in burn patients.     

GSK-3beta activity is increased in skeletal muscle after burn injury in rats

Previous reports suggest that burn-induced muscle proteolysis can be inhibited by treatment with GSK-3beta inhibitors, suggesting that burn injury may be associated with increased GSK-3beta activity. The influence of burn injury on muscle GSK-3beta activity, however, is not known. We determined the effect of a 30% total body surface full-thickness burn injury in rats on muscle GSK-3beta activity by measuring GSK-3beta activity and tissue levels of serine 9 phosphorylated GSK-3beta, p(Ser9)-GSK-3beta, by Western blot analysis and immunohistochemistry. Because burn-induced muscle wasting is, at least in part, mediated by glucocorticoids, we used dexamethasone-treated cultured muscle cells in which GSK-3beta expression was reduced with small interfering RNA (siRNA) to further assess the role of GSK-3beta in muscle atrophy. Burn injury resulted in a seven-fold increase in GSK-3beta activity in skeletal muscle. This effect of burn was accompanied by reduced tissue levels of p(Ser9)-GSK-3beta, suggesting that burn injury stimulates GSK-3beta in skeletal muscle secondary to inhibited phosphorylation of the enzyme. In addition, burn injury resulted in inhibited phosphorylation and activation of Akt, an upstream regulatory mechanism of GSK-3beta activity. Reducing the expression of GSK-3beta in cultured muscle cells with siRNA inhibited dexamethasone-induced protein degradation by approximately 50%. The results suggest that burn injury stimulates GSK-3beta activity in skeletal muscle and that GSK-3beta may, at least in part, regulate glucocorticoid-mediated muscle wasting.     

Myosin light chain kinase mediates intestinal barrier disruption following burn injury

Background

Severe burn injury results in the loss of intestinal barrier function, however, the underlying mechanism remains unclear. Myosin light chain (MLC) phosphorylation mediated by MLC kinase (MLCK) is critical to the pathophysiological regulation of intestinal barrier function. We hypothesized that the MLCK-dependent MLC phosphorylation mediates the regulation of intestinal barrier function following burn injury, and that MLCK inhibition attenuates the burn-induced intestinal barrier disfunction.

Methodology/Principal Findings

Male balb/c mice were assigned randomly to either sham burn (control) or 30% total body surface area (TBSA) full thickness burn without or with intraperitoneal injection of ML-9 (2 mg/kg), an MLCK inhibitor. In vivo intestinal permeability to fluorescein isothiocyanate (FITC)-dextran was measured. Intestinal mucosa injury was assessed histologically. Tight junction proteins ZO-1, occludin and claudin-1 was analyzed by immunofluorescent assay. Expression of MLCK and phosphorylated MLC in ileal mucosa was assessed by Western blot. Intestinal permeability was increased significantly after burn injury, which was accompanied by mucosa injury, tight junction protein alterations, and increase of both MLCK and MLC phosphorylation. Treatment with ML-9 attenuated the burn-caused increase of intestinal permeability, mucosa injury, tight junction protein alterations, and decreased MLC phosphorylation, but not MLCK expression.

Conclusions/Significance

The MLCK-dependent MLC phosphorylation mediates intestinal epithelial barrier dysfunction after severe burn injury. It is suggested that MLCK-dependent MLC phosphorylation may be a critical target for the therapeutic treatment of intestinal epithelial barrier disruption after severe burn injury.     

Hyperglycemia Exacerbates Burn-Induced Liver Inflammation via Noncanonical Nuclear Factor-κB Pathway Activation

Hyperglycemia and inflammation are hallmarks of burn injury. In this study, we used a rat model of hyperglycemia and burn injury to investigate the effects of hyperglycemia on inflammatory responses in the liver. Hyperglycemia was induced in male Sprague-Dawley rats with streptozotocin (STZ) (35–40 mg/kg), followed by a 60% third-degree scald burn injury. Cytokine levels (by multiplex, in cytosolic liver extracts), hormones (by enzyme-linked immunosorbent assay [ELISA], in serum), nuclear factor (NF)-κB protein deoxyribonucleic acid (DNA) binding (by ELISA, in nuclear liver extracts) and liver functional panel (using VetScan, in serum) were measured at different time points up to 7 d after burn injury. Blood glucose significantly increased after burn injury in both groups with different temporal patterns. Hyperglycemic rats were capable of endogenous insulin secretion, which was enhanced significantly versus controls 12 h after burn injury. DNA binding data of liver nuclear extracts showed a robust and significant activation of the noncanonical NF-κB pathway in the hyperglycemic versus control burn animals, including increased NF-κB–inducing kinase expression (p < 0.05). Liver acute-phase proteins and cytokine expression were increased, whereas secretion of constitutive proteins was decreased after burn injury in hyperglycemic versus control animals (p < 0.05). These results indicate that burn injury to the skin rapidly activated canonical and noncanonical NF-κB pathways in the liver. Robust activation of the NF-κB noncanonical pathway was associated with increased expression of inflammatory markers and acute-phase proteins, and impaired glucose metabolism. Hyperglycemia is detrimental to burn outcome by augmenting inflammation mediated by hepatic noncanonical NF-κB pathway activation.     

Systemic inflammation and remote organ damage following bilateral femur fracture requires Toll-like receptor 4

Extensive soft tissue injury and bone fracture are significant contributors to the initial systemic inflammatory response in multiply injured patients. Systemic inflammation can lead to organ dysfunction remote from the site of traumatic injury. The mechanisms underlying the recognition of peripheral injury and the subsequent activation of the immune response are unknown. Toll-like receptors (TLRs) recognize microbial products but also may recognize danger signals released from damaged tissues. Here we report that peripheral tissue trauma initiates systemic inflammation and remote organ dysfunction. Moreover, this systemic response to a sterile local injury requires toll-like receptor 4 (TLR4). Compared with wild-type (C3H/HeOuJ) mice, TLR4 mutant (C3H/HeJ) mice demonstrated reduced systemic and hepatic inflammatory responses to bilateral femur fracture. Trauma-induced nuclear factor (NF)-kappaB activation in the liver required functional TLR4 signaling. CD14-/- mice failed to demonstrate protection from fracture-induced systemic inflammation and hepatocellular injury. Therefore, our results also argue against a contribution of intestine-derived LPS to this process. These findings identify a critical role for TLR4 in the rapid recognition and response pathway to severe traumatic injury. Application of these findings in an evolutionary context suggests that multicellular organisms have evolved to use the same pattern recognition receptor for surviving traumatic and infectious challenges.     

Molecular mechanism(s) of burn-induced insulin resistance in murine skeletal muscle: role of IRS phosphorylation

Hyperglycemia, glucose intolerance and elevated insulin levels frequently occur in burned patients; however, the mechanism(s) for this insulin resistance has not been fully elucidated. One possible mechanism could involve alterations in the phosphorylation of serine 307 of the insulin receptor substrate-1 (IRS-1) via activation of stress kinase enzymes, including SAPK/JNK. In the present study we examined the time course of the effect of burn injury to mice on: levels of IRS-1 protein, phosphorylation of serine 307 of IRS-1, SAPK/JNK kinase levels and activity and Akt kinase activity in hind limb skeletal muscle. Burn injury produced a reduction in hind limb muscle mass 24 h after injury, and, which persisted for 168 h. At 24 h after injury, there was a dramatic ( approximately 9-fold) increase in phosphorylation of IRS-1 serine 307 followed by a more moderate elevation thereafter. Total IRS-1 protein was slightly elevated at 24 h after injury and decreased to levels below sham treated animals at the later times. Burn injury did not appear to change total SAPK/JNK protein content, however, enzyme activity was increased for 7 days after injury. Akt kinase activity was decreased in skeletal muscle following burn injury; providing a biochemical basis for burn-induced insulin resistance. These findings are consistent with the hypothesis that burn-induced insulin resistance may be related, at least in part, to alterations in the phosphorylation of key proteins in the insulin signaling cascade, including IRS-1, and that changes in stress kinases, such as SAPK/JNK produced by burn injury, may be responsible for these changes in phosphorylation.     

Recombinant plasma gelsolin infusion attenuates burn-induced pulmonary microvascular dysfunction.

Reduced plasma concentrations of the extracellular actin-binding proteins gelsolin and Gc-globulin correlate with pulmonary failure and death in humans after injury. The purpose of this study was to investigate the role of plasma gelsolin in the pathophysiology of inflammation-induced lung injury. We postulated that plasma gelsolin levels decrease at an early time point after burn injury and that the intravenous infusion of gelsolin prevents burn-induced pulmonary microvascular dysfunction. Adult Sprague-Dawley rats were randomized to undergo a 40% body surface area thermal injury (Burn) or manipulation without burn (Sham). Plasma gelsolin and Gc-globulin concentrations were determined at various times during the first 6 days of injury by Western blotting. Other animals were randomized to receive either recombinant human gelsolin (0.078, 0.78, or 7.8 mg) or albumin (7.8 mg) before and 8 h after Burn or Sham. Twenty-four hours later, pulmonary microvascular permeability was assessed by measuring the capillary filtration by use of an isolated, perfused lung model. We found that plasma gelsolin levels of burn-injured rats decreased to 10% of normal levels within 12 h and remained below normal levels for up to 6 days postinjury. Gc-globulin values also fall, but to a lesser extent and only transiently. Treatment of burned animals with intravenous infusions of recombinant human gelsolin prevented the increase in pulmonary microvascular permeability that accompanies this injury. Our findings are consistent with the hypothesis that plasma gelsolin depletion contributes to the pathophysiology of pulmonary microvascular dysfunction during inflammation.     

Burn-induced oxidative injury of the gut is ameliorated by the leukotriene receptor blocker montelukast

There is increasing evidence that oxidative stress has an important role in the development of multiorgan failure after major burn injury. In the present study, we investigated whether the leukotriene receptor blocker montelukast is protective against burn-induced injury of the gut. Under brief ether anaesthesia, shaved dorsum of the rats was exposed to 90 degrees C (burn group) or 25 degrees C (control group) water bath for 10 s. Montelukast (10 mg/kg) or saline was administered intraperitoneally immediately after and at the 12th hour of the burn injury. Rats were decapitated 24 h after burn injury and the skin samples, as well as tissue samples from stomach, ileum and colon, were taken for the determination of malondialdehyde (MDA) and glutathione (GSH) levels, myeloperoxidase (MPO) activity and collagen contents. Tissues were also examined microscopically. Tumor necrosis factor-alpha (TNF-alpha) and lactate dehydrogenase (LDH) were assayed in serum samples. Severe skin scald injury (30% of total body surface area) caused a significant decrease in GSH level, which was accompanied with significant increases in MDA level, MPO activity and collagen content of tissues. Similarly, serum TNF-alpha and LDH were elevated in the burn group as compared to control group. On the other hand, montelukast treatment reversed all these biochemical indices, as well as histopathological alterations, which were induced by thermal trauma. Findings of the present study suggest that montelukast possesses an anti-inflammatory effect on burn-induced gastrointestinal damage and protects against oxidative injury by a neutrophil-dependent mechanism.     

In vivo rabbit hindquarter model for assessment of regional burn hypermetabolism.

Severe burn injury evokes hypermetabolism and muscle wasting, despite nominally adequate nutrition. Although there is much information on whole organism and isolated tissue metabolism after burn injury, data examining regional burn hypermetabolism in vivo are lacking. Using surgically implanted (general anesthesia) regional vascular catheters and primed constant infusion of l-[1-(13)C]phenylalanine tracer, we have determined in vivo burn-induced alterations in rabbit hindquarter protein and energy metabolism. Burn injury evokes increased whole body resting energy expenditure and phenylalanine turnover, accompanied by significantly increased hindquarter proteolysis, creating a negative protein balance in burned rabbit hindquarter. Hindquarter oxygen consumption showed an increase after burn injury, but it did not reach statistical significance. Burn-induced changes in hindquarter protein turnover account for approximately one-third of the whole animal hypermetabolism. This model offers a system for regional manipulation of postburn hypermetabolism.     

Octreotide improves burn-induced intestinal injury in the rat

The local thermal trauma activates a number of systemic mediator cascades, e.g. a complement activation, cytokine production, resulting in a generalized sequestration and a priming of local and systemic neutrophils and macrophages. We aimed to determine the possible protective effect of octreotide (OCT), a synthetic somatostatin analogue, against burn-induced intestinal tissue damage possibly by inhibiting neutrophil infiltration. Under brief ether anaesthesia, shaved dorsum of the rats was exposed to 90 degrees C bath for 10s to induce burn injury. Rats were decapitated either 3, 24 or 72 h after burn injury. Octreotide (10 microg/kg) or saline was administered subcutaneously (s.c.) immediately after the burn injury. In the 24- and 72-h burn groups, OCT injections were repeated three times daily. In the sham group the same protocol was applied except that the dorsum was dipped in a 25 degrees C water bath for 10 s Malondialdehyde (MDA) and glutathione (GSH) levels and myeloperoxidase (MPO) activity were determined in the intestinal tissue. The results demonstrate that burn injury results in significant neutrophil accumulation, as evidenced by increases in MPO activity. The increase in MDA and the concomitant decrease in GSH levels demonstrate the role of oxidative mechanisms in burn injury. OCT may have some beneficial therapeutic effects by reducing neutrophil-dependent injury and related lipid peroxidation following burn trauma.