Burned Mice Research Articles

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Reply We are responding to concerns raised in this Letter to the Editor that the solvent used during delivery of the specific caspase 1 inhibitor, AC-YVAD-cmk, to mice may have contributed to the observation that caspase 1 inhibition significantly increased the mortality in burn mice. We understand their concerns but wish to assure them and other readers that the concentration of solvent used in our studies was significantly lower than what has been reported to cause inflammation and increases in immune cell apoptosis in mice. The referenced study used 8 mg/kg of dimethyl sulfoxide (DMSO) before significant changes in immune cell apoptosis were observed (1). The amount of DMSO given with our AC-YVAD-cmk treatment was 0.5 mg/kg (2). This is 16-fold less than what was reported to cause apoptosis in mice. Other studies using DMSO to study changes in inflammatory cytokine production were done in vitro or at higher doses than what we used to deliver AC-YVAD-cmk (1, 3). Also, the stimulus used to induce inflammation was bacterial lipopolysaccharide (LPS), and the inflammatory reactions caused by LPS and trauma are not similar. In addition, one of the cytokines reported to be induced by DMSO exposure and LPS was interleukin 1β (IL-1β) (3). It is important to restate in this letter that we clearly showed that giving AC-YVAD-cmk in DMSO potently blocked injury-induced IL-1β production in vivo (2). Moreover, we performed in vivo dosing studies to demonstrate that giving AC-YVAD-cmk in DMSO to mice at doses that specifically reduced injury-induced caspase 1 activation led to significant mortality in burn mice. Finally, in control experiments comparing saline to vehicle (0.5 mg/kg DMSO), we found no measurable effect on mortality (n = 10 mice/group over 7 days) or systemic levels of injury-induced IL-6 production. We thank them for their opinions and interest in our research report. In closing, based on our findings, we are currently expanding our efforts to determine how inflammasome activation by injury protects mice from injury-induced complications. We recently found that caspase 1−/− mice also showed an enhanced inflammatory phenotype at 1 day after injury. Furthermore, a recent study using caspase 1−/− mice confirmed our findings showing that that caspase 1 was protective in a trauma mouse model of hemorrhagic shock with combined femur fracture (4). Thus, we are keenly interested in determining if stimulating inflammasome or caspase 1 activation could be used to protect from the acute complications of traumatic injuries. Akinori Osuka Marc Hanschen Veit Stoecklein James Lederer Brigham and Women’s Hospital Biomedical Research Institute and Harvard Medical School Boston, Massachusetts

Optimization and application of a flow cytometric PU.1 assay for murine immune cells

PU.1 is a master transcription factor whose levels directly influence hematopoiesis, leukemia, susceptibility to sepsis, and macrophage function. Though measurement of PU.1 levels is important to health and disease, most studies have relied on PCR or western blots to measure the expression of this transcription factor. An accessible, validated assay that could measure PU.1 protein in subpopulations of cells is needed. In this work we present an optimized flow cytometric assay to detect PU.1 in subpopulations of immune cells. Murine myeloid cells were fixed in paraformaldehyde, permeabilized, and then stained with anti PU.1 in the presence and absence of a blocking peptide containing the binding site of the antibody. The bound anti PU.1 was then visualized with a labeled second antibody. Methanol and ethanol were tested for their relative ability to permeabilize cells and detect PU.1. The effect of the procedure upon the ability to detect cellular subpopulations was examined. Relative PU.1 1evels in normal T cells, B cells, monocytes, macrophages, dendritic cells, neutrophils, and progenitors from the spleen and/or bone marrow were determined. Finally, PU.1 levels in proliferating myeloid cells from burn mice were determined. There was a dose dependent increase in the amount of PU.1 detected with increasing amounts of PU.1 antibody that was not seen when blocking peptide was used. Methanol or ethanol gave equivalent results as permeabilization agents, but the latter allowed easier detection of surface antigens when surface staining was performed prior to permeabilization. T cells had little if any PU.1, while B cells had intermediate levels of PU.1, and myeloid cells had high levels of PU.1. Monocytes had higher levels of PU.1 than did neutrophils or spleen macrophages. Plasmacytoid dendritic cells had lower levels of PU.1 than did conventional dendritic cells. Immature myeloid cells had higher levels of PU.1 than did mature myeloid cells. In addition, PU.1 levels were higher in proliferating cells than the corresponding non proliferating cells. Myeloid cells derived from burn mice tended to have higher levels of PU.1 than did unburned, but proliferating cells from burn or sham mice showed no difference in their levels of PU.1. This assay should be a useful addition to the tools used to study the function of PU.1 in health and disease.

Pseudomonas aeruginosa infection after burn injury polarizes innate immune cells towards an alternatively activated phenotype (55.19)

Abstract Severe burn injury causes profound immunosuppression resulting in prevalent bacterial infections. We hypothesized that bacterial infection after burn injury polarizes macrophage (Mo) and neutrophil (No) responses into an alternatively activated (M2/N2) phenotype. To test this, we used a murine model of burn injury and bacterial challenge. Female C57BL/6 mice underwent either a 20% TBSA full thickness contact burn or control sham procedure. Twenty-four hours later mice were either given a subcutaneous injection of Pseudomonas aeruginosa or control saline and sacrificed two days later to determine bacterial burden in skin, liver and lung. Serum cytokine levels were measured by cytometric bead array. Splenocytes were cultured in the presence of Brefeldin-A for intracellular cytokine analysis, and Mo and No were identified by surface staining. Burn mice had bacterial dissemination indicated by positive cultures from all tissue samples. No bacteria were recovered from sham mice. Burn injury, but not infection, induced a significant increase in serum IFN-γ and TNF-α. IL-6 was also significantly increased after burn injury alone but was significantly higher in infected burn mice. IL-10 was elevated in infected burn mice only. Flow cytometry confirmed expression of IL-10 protein within Mo and No. These data suggest that after burn injury, bacterial infection polarizes serum cytokine and innate cell phenotype into an alternatively activated phenotype allowing systemic infection.

Antimicrobial Photodynamic Therapy on Drug‐resistant Pseudomonas aeruginosa‐induced Infection. An In Vivo Study†

Pseudomonas aeruginosa is considered one of the most important pathogens that represent life-threatening risk in nosocomial environments, mainly in patients with severe burns. Antimicrobial photodynamic therapy (aPDT) has been effective to kill bacteria. The purpose of this study was to develop a burn wound and bloodstream infection model and verify aPDT effects on it. In vitro, we tested two wavelengths (blue and red LEDs) on a clinical isolate of P. aeruginosa strain with resistance to multiple antibiotics using HB:La(+3) as photosensitizer. Verapamil(®) associated to aPDT was also studied. In vivo, P. aeruginosa-infected burned mice were submitted to aPDT. Bacterial counting was performed on local infection and bloodstream. Survival time of animals was also monitored. In this study, aPDT was effective to reduce P. aeruginosa in vitro. In addition, Verapamil(®) assay showed that HB:La(+3) is not recognized by ATP-binding cassete (ABC) efflux pump mechanism. In the in vivo study, aPDT was able to reduce bacterial load in burn wounds, delay bacteremia and keep the bacterial levels in blood 2-3 logs lower compared with an untreated group. Mice survival was increased on 24 h. Thus, this result suggests that aPDT may also be a novel prophylactic treatment in the care of burned patients.

Inflammation, organomegaly, and muscle wasting despite hyperphagia in a mouse model of burn cachexia

BackgroundBurn injury results in a chronic inflammatory, hypermetabolic, and hypercatabolic state persisting long after initial injury and wound healing. Burn survivors experience a profound and prolonged loss of lean body mass, fat mass, and bone mineral density, associated with significant morbidity and reduced quality of life. Understanding the mechanisms responsible is essential for developing therapies. A complete characterization of the pathophysiology of burn cachexia in a reproducible mouse model was lacking.MethodsYoung adult (12–16 weeks of age) male C57BL/6J mice were given full thickness burns using heated brass plates or sham injury. Food and water intake, organ and muscle weights, and muscle fiber diameters were measured. Body composition was determined by Piximus. Plasma analyte levels were determined by bead array assay.ResultsSurvival and weight loss were dependent upon burn size. The body weight nadir in burned mice was 14 days, at which time we observed reductions in total body mass, lean carcass mass, individual muscle weights, and muscle fiber cross-sectional area. Muscle loss was associated with increased expression of the muscle ubiquitin ligase, MuRF1. Burned mice also exhibited reduced fat mass and bone mineral density, concomitant with increased liver, spleen, and heart mass. Recovery of initial body weight occurred at 35 days; however, burned mice exhibited hyperphagia and polydipsia out to 80 days. Burned mice had significant increases in serum cytokine, chemokine, and acute phase proteins, consistent with findings in human burn subjects.ConclusionsThis study describes a mouse model that largely mimics human pathophysiology following severe burn injury. These baseline data provide a framework for mouse-based pharmacological and genetic investigation of burn-injury-associated cachexia.

Prior Thermal Injury Accelerates Endotoxin-Induced Inflammatory Cytokine Production and Intestinal Nuclear Factor-κB Activation in Mice

The objective of this study was to increase the understanding of the "second-hit" response in thermal injury. The authors hypothesized that prior thermal injury increases the endotoxin-induced inflammatory response of intestinal mucosa. Mice underwent sham or 25% TBSA scald injury. Seven days after injury, mice were injected with lipopolysaccharide. Blood, jejunum, and colon specimens were obtained at intervals. Serum, jejunal, and colon inflammatory cytokine levels were measured by enzyme-linked immunosorbent assay. Jejunal and colon nuclear factor (NF)-κB activation was measured by electrophoretic mobility shift assay. After remote thermal injury, lipopolysaccharide exposure led to an acute increase in serum interleukin (IL)-6, IL-10, and chemokine keratinocyte-derived chemokine (KC) levels. This correlated with lipopolysaccharide-induced increased IL-6 in colon and chemokine KC in the jejunum and colon in burned mice when compared with sham-injured mice. Lipopolysaccharide-induced NF-κB activation occurred more rapidly in jejunum and colon from burned mice compared with sham-injured mice. Prior thermal injury accelerates lipopolysaccharide-induced inflammatory cytokine production systemically in jejunum and colon. The "second hit" of lipopolysaccharide led to earlier intestinal NF-κB activation in burned mice compared with sham-injured mice. These results indicate that there is a heightened inflammatory response by jejunum and colon in response to a "second hit" of lipopolysaccharide after burn injury.

Effect of IL-10 antisense gene therapy in severely burned mice intradermally infected with MRSA

The effect of IL-10 antisense oligodeoxynucleotides (ODN) on the susceptibility of burned mice to intradermal (i.d.) infection of methicillin-resistant Staphylococcus aureus (MRSA) was studied. Abscesses formed and sepsis did not develop in normal mice infected i.d. with 108CFU/mouse of MRSA. Similarly, sepsis caused by MRSA i.d. infection did not develop and abscesses formed in burned mice treated with IL-10 antisense ODN. However, all of the burned mice treated with scrambled ODN (control group) died by infectious complications stemming from MRSA i.d. infection, and an MRSA-abscess did not form in these mice. Macrophages (Mϕ) isolated from the infection site tissue of burned mice that were treated with IL-10 antisense ODN were identified as M1Mϕ, while Mϕ isolated from burned mice that were treated with scrambled ODN were shown to be M2Mϕ. MRSA-abscesses formed in burned mice inoculated with M1Mϕ, and these mice resisted a lethal dose of MRSA i.d. infection. However, an abscess did not form, and sepsis caused by MRSA i.d. infection developed in burned mice that were inoculated with M2Mϕ. These results indicate that severely burned mice treated with IL-10 antisense ODN are resistant against i.d. infection with MRSA. M1Mϕ appeared in the infection site tissues of severely burned mice that were treated with IL-10 antisense ODN may play a role on the abscess formation and inhibiting sepsis caused by MRSA i.d. infection.

Effect of CCL2 antisense oligodeoxynucleotides on bacterial translocation and subsequent sepsis in severely burned mice orally infected with Enterococcus faecalis

Severely burned mice are susceptible to sepsis stemming from Enterococcus faecalis translocation due to the impaired generation of M1 macrophages (M1MΦs) in local translocation sites. In our previous studies, CCL2 has been characterized as a major effector molecule on the burn-associated generation of M2MΦs, an inhibitor cell type for resident MΦ conversion into M1MΦs. In this study, we tried to protect burned mice orally infected with E. faecalis utilizing CCL2 antisense oligodeoxynucleotides (ODNs). We show that M2MΦs in mesenteric lymph nodes (MLNs) were not demonstrated in burned mice treated with CCL2 antisense ODNs. M1MΦs were not induced by heat-killed E. faecalis from resident MΦs transwell-cultured with mesenteric lymph node macrophages (MLN-MΦs) from burned mice, while M1MΦs were induced by the same antigen from resident MΦs transwell-cultured with MΦs which were isolated from burned mice treated with CCL2 antisense ODNs. Bacterial growth in MLNs was shown in burned mice orally infected with a lethal dose of E. faecalis. However, after the same infection, sepsis did not develop in burned mice treated with CCL2 antisense ODNs. These results indicate that bacterial translocation and subsequent sepsis are controlled in burned mice orally infected with a lethal dose of E. faecalis by gene therapy utilizing CCL2 antisense ODNs.

Novel Mitochondria-Targeted Antioxidant Peptide Ameliorates Burn-Induced Apoptosis and Endoplasmic Reticulum Stress in the Skeletal Muscle of Mice

This study tested the hypothesis that a novel mitochondria-targeted SS-31 peptide attenuates the burn injury-induced apoptosis and endoplasmic reticulum stress and improves insulin sensitivity in the skeletal muscle. Following 30% total body surface area burn or sham burn, mice were injected daily with SS-31 peptide (5 mg/kg body weight), and the rectus abdominis muscles collected on postburn days 1, 3, and 7. The tissues were subjected to various biochemical and immunohistochemical analyses. Treatment with SS-31 peptide prevented burn-induced increases in the caspase 3 activity (P < 0.05) and apoptosis (P < 0.01) on postburn day 7. The SS-31 peptide treatment also prevented the increase in the expression levels of phosphatase and tensin homolog on postburn days 3 and 7. Burn injury-induced increases in the levels of two endoplasmic reticulum stress markers, binding immunoglobulin protein and protein disulfide isomerase, were significantly decreased by the SS-31 peptide treatments on postburn day 7 and on day 3 for binding immunoglobulin protein as well (P < 0.05). The effects of SS-31 appear to be, in part, due to its ability to reduce oxidative stress in burned mice, evidenced by reduced expression of oxidized proteins that were clearly evident on postburn day 7. Our results demonstrate a possible therapeutic potential of SS-31 peptide to ameliorate the adverse effects of burn injury in skeletal muscle.

What’s New in Shock, December 2011?

What’s New in Shock, December 2011?

High MafB expression following burn augments monocyte commitment and inhibits DC differentiation in hemopoietic progenitors

We have previously shown that perturbed bone marrow progenitor development promotes hyporesponsive monocytes following experimental burn sepsis. Clinical and experimental sepsis is associated with monocyte deactivation and depletion of mDCs. Decrease in circulating DCs is reported in burn patients who develop sepsis. In our 15% TBSA scald burn model, we demonstrate a significant reduction in the circulating MHC-II(+) population and mDCs (Gr1(neg)CD11b(+)CD11c(+)) with a corresponding decrease in bone marrow MHC-II(+) cells and mDCs for up to 14 days following burn. We explored the underlying mechanism(s) that regulate bone marrow development of monocytes and DCs following burn injury. We found a robust bone marrow response with a significant increase in multipotential HSCs (LSK) and bipotential GMPs following burn injury. GMPs from burn mice exhibit a significant reduction in GATA-1, which is essential for DC development, but express high levels of MafB and M-CSFRs, both associated with monocyte production. GMPs obtained from burn mice differentiated 1.7 times more into Mϕ and 1.6-fold less into DCs compared with sham. Monocytes and DCs expressed 50% less MHC-II in burn versus sham. Increased monocyte commitment in burn GMPs was a result of high MafB and M-CSFR expressions. Transient silencing of MafB (siRNA) in GMP-derived monocytes from burn mice partially restored DC differentiation deficits and increased GATA-1 expression. We provide evidence that high MafB following burn plays an inhibitory role in monocyte-derived DC differentiation by regulating M-CSFR and GATA-1 expressions.

Treatment with gelsolin reduces brain inflammation and apoptotic signaling in mice following thermal injury

BackgroundBurn survivors develop long-term cognitive impairment with increased inflammation and apoptosis in the brain. Gelsolin, an actin-binding protein with capping and severing activities, plays a crucial role in the septic response. We investigated if gelsolin infusion could attenuate neural damage in burned mice.MethodsMice with 15% total body surface area burns were injected intravenously with bovine serum albumin as placebo (2 mg/kg), or with low (2 mg/kg) or high doses (20 mg/kg) of gelsolin. Samples were harvested at 8, 24, 48 and 72 hours postburn. The immune function of splenic T cells was analyzed. Cerebral pathology was examined by hematoxylin/eosin staining, while activated glial cells and infiltrating leukocytes were detected by immunohistochemistry. Cerebral cytokine mRNAs were further assessed by quantitative real-time PCR, while apoptosis was evaluated by caspase-3. Neural damage was determined using enzyme-linked immunosorbent assay of neuron-specific enolase (NSE) and soluble protein-100 (S-100). Finally, cerebral phospho-ERK expression was measured by western blot.ResultsGelsolin significantly improved the outcomes of mice following major burns in a dose-dependent manner. The survival rate was improved by high dose gelsolin treatment compared with the placebo group (56.67% vs. 30%). Although there was no significant improvement in outcome in mice receiving low dose gelsolin (30%), survival time was prolonged against the placebo control (43.1 ± 4.5 h vs. 35.5 ± 5.0 h; P < 0.05). Burn-induced T cell suppression was greatly alleviated by high dose gelsolin treatment. Concurrently, cerebral abnormalities were greatly ameliorated as shown by reduced NSE and S-100 content of brain, decreased cytokine mRNA expressions, suppressed microglial activation, and enhanced infiltration of CD11b+ and CD45+ cells into the brain. Furthermore, the elevated caspase-3 activity seen following burn injury was remarkably reduced by high dose gelsolin treatment along with down-regulation of phospho-ERK expression.ConclusionExogenous gelsolin infusion improves survival of mice following major burn injury by partially attenuating inflammation and apoptosis in brain, and by enhancing peripheral T lymphocyte function as well. These data suggest a novel and effective strategy to combat excessive neuroinflammation and to preserve cognition in the setting of major burns.

Inducible nitric oxide synthase deficiency ameliorates skeletal muscle insulin resistance but does not alter unexpected lower blood glucose levels after burn injury in C57BL/6 mice

Burn injury is associated with inflammatory responses and metabolic alterations including insulin resistance. Impaired insulin receptor substrate-1 (IRS-1)–mediated insulin signal transduction is a major component of insulin resistance in skeletal muscle following burn injury. To further investigate molecular mechanisms that underlie burn injury–induced insulin resistance, we study a role of inducible nitric oxide synthase (iNOS), a major mediator of inflammation, on burn-induced muscle insulin resistance in iNOS-deficient mice. Full-thickness third-degree burn injury comprising 12% of total body surface area was produced in wild-type and iNOS-deficient C57BL/6 mice. Insulin-stimulated activation (phosphorylation) of IR, IRS-1, and Akt was assessed by immunoblotting and immunoprecipitation. Insulin-stimulated glucose uptake by skeletal muscle was evaluated ex vivo. Burn injury caused induction of iNOS in skeletal muscle of wild-type mice. The increase of iNOS expression paralleled the increase of insulin resistance, as evidenced by decreased tyrosine phosphorylation of IR and IRS-1, IRS-1 expression, insulin-stimulated activation of phosphatidylinositol 3-kinase and Akt/PKB, and insulin-stimulated glucose uptake in mouse skeletal muscle. The absence of iNOS in genetically engineered mice significantly lessened burn injury–induced insulin resistance in skeletal muscle. In wild-type mice, insulin tolerance test revealed whole-body insulin resistance in burned mice compared with sham-burned controls. This effect was reversed by iNOS deficiency. Unexpectedly, however, blood glucose levels were depressed in both wild-type and iNOS-deficient mice after burn injury. Gene disruption of iNOS ameliorated the effect of burn on IRS-1–mediated insulin signaling in skeletal muscle of mice. These findings indicate that iNOS plays a significant role in burn injury–induced skeletal muscle insulin resistance.

Neutrophils, Not Monocyte/Macrophages, are the Major Splenic Source of Postburn IL-10

Burn induces myeloid-derived suppressor cells (MDSCs), a heterogeneous population of immature polymorphonuclear neutrophils (PMNs) and monocytes, which protect against infection. Previous work from our laboratory demonstrated that inflammatory monocytes (iMos) were the major MDSC source of TNF-α in the postburn spleen, and we hypothesized that they were also the major source of postburn IL-10. To test this hypothesis, we examined cytokine production by postburn CCR2 knockout (KO) mice, which have fewer iMos than burn wild-type (WT) splenocytes, but equal numbers of PMNs and F4/80 macrophages. Using cell sorting and/or intracellular cytokine techniques, we examined IL-10 production by postburn PMNs and iMos. Finally, we compared IL-10 production by postburn PMNs and iMos with culture-derived MDSCs. Splenocytes from postburn CCR2 KO mice produced less IL-6 and TNF-α than WT burn splenocytes in response to LPS, but KO and WT burn splenocytes produced equal amounts of IL-10 in response to peptidoglycan. Depletion of PMNs from postburn splenocytes led to reductions in IL-10 and increases in IL-6 and TNF-α in response to peptidoglycan, but not in response to LPS. Sorting or intracellular cytokine techniques gave consistent results: Burn PMNs made more IL-10 than sham PMNs and also more IL-10 than burn or sham iMos. Polymorphonuclear neutrophil and iMos subpopulations from culture-derived MDSCs produced the same cytokine profiles in response to LPS and peptidoglycan as did the PMNs and iMos from postburn spleens: PMNs made IL-10, whereas iMos made IL-6. Finally, LPS-induced mortality of burn mice was made worse by anti-Gr-1 depletion of all PMNs and 66% of iMos from burn mice. This suggests that PMNs play a primarily anti-inflammatory role in vitro and in vivo.

Evaluation of the antioxidant peptide SS31 for treatment of burn-induced insulin resistance

After severe burn injury and other major traumas, glucose tolerance tests demonstrate delayed glucose disposal. This 'diabetes of injury' could be explained by insulin deficiency, and several studies have shown that soon after trauma (ebb phase) insulin concentrations are reduced in the face of hyperglycemia. After resuscitation of trauma patients (flow phase), β-cell responsiveness normalizes and plasma insulin levels are appropriate or even higher than expected, however, glucose intolerance and hyperglycemia persist. In the acute care setting, several approaches have been used for treating insulin resistance, including insulin infusion, propranolol and glucagon-like-peptide-1 (GLP-1). Recently, it was demonstrated that a tetrapeptide with antioxidant properties D-Arg-Dmt-Lys-Phe-NH2 (SS31), but not its inactive analogue Phe-D-Arg-Phe-Lys-NH2 (SS20) attenuates insulin resistance in mice maintained on a high fat diet. In this report the effects of SS31 and SS20 on burn-induced insulin resistance was studied in mice. Oral glucose tolerance tests (OGTT) were performed in 4 groups of 6 mice with thermal injury with or without pre-treatment with SS31 or SS20 and sham controls. In addition, biodistribution of 18FDG was measured in burned mice with and without SS31 treatment and shams (subsets of these animals were also studied by µPET). For comparison purposes, groups of 6 cold-stressed mice with and without SS31 treatment were also studied. The results of these studies demonstrate that SS31 but not SS20 ameliorated burn-induced insulin resistance. In addition, SS31 treatment resulted in marked reduction in the increased 18FDG uptake by brown adipose tissue (BAT) in burned but not cold-stressed animals; suggesting that the stressors act by different mechanisms. Overall, these studies confirmed that SS31 can be used to reverse burn-induced insulin resistance and provide a firm pre-clinical basis for future clinical trials of SS31 for the treatment of insulin resistance in patients with burn injury.

Neutralization effects of egg yolk immunoglobulin (IgY) and Fab' fragment against lipopolysaccharide (LPS) in burned mice model

The aim of this study is to evaluate the neutralization effects of egg yolk immunoglobulin (IgY) and Fab’ fragment against lipopolysaccharide (LPS), and identify possible approaches to prevent and treat LPS related injuries. Mice with third-degree burns covering 30% of the total body surface and exposed to LPS were orally administered with the IgY or Fab’ fragment of egg yolk immunoglobulin at 6, 12, 24 and 48 h after burning. The mortality rates and circulating levels of LPS, tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) were determined. The mortality rate of mice in the IgY-treated groups and Fab’treated groups were significantly lower when compared with the control groups (without treatment). The levels of LPS, TNF-α and IL-6 in the IgY-treated group and Fab’-treated group were lower than those in the control group, and Fab’-treated group posses much lower levels of LPS, TNF-α and IL-6 than IgYtreated group. The IgY and Fab’ fragment of egg yolk immunoglobulin raised against LPS may be used to inhibit and treat tissue injury caused by LPS. IgY technology has significant future opportunities for the prevention and treatment of LPS related infectious gastrointestinal diseases in humans and animals.

Burn-induced alterations in toll-like receptor-mediated responses by bronchoalveolar lavage cells

Burn is associated with profound inflammation and activation of the innate immune system in multiple organ beds, including the lung. Similarly, toll-like receptors (TLR) are associated with innate immune activation. Nonetheless, it is unclear what impact burn has on TLR-induced inflammatory responses in the lung. Methods Male C57BL/6 mice were subjected to burn (3rd degree, 25% TBSA) or sham procedure and 1, 3 or 7 days thereafter, bronchoalveolar lavage (BAL) fluid was collected and cells were isolated and cultured in vitro with specific TLR agonists as follows: Zymosan (TLR-2), LPS (TLR-4) and CpG-ODN (TLR-9). Supernatants were collected 48 h later and assayed for inflammatory cytokine levels (IL-1β, IL-6, IL-10, IL-17, TNF-α, KC, MCP-1, MIP-1α, MIP-1β and RANTES) by Bioplex. Results BAL fluid from sham and burn mice did not contain detectable cytokine levels. BAL cells, irrespective of injury, were responsive to TLR-2 and TLR-4 activation. Seven days after burn, TLR-2 and TLR-4 mediated responses by BAL cells were enhanced as evidenced by increased production of IL-6, IL-17, TNF-α, MCP-1, MIP-1β and RANTES. Conclusions Burn-induced changes in TLR-2 and TLR-4 reactivity may contribute to the development of post-burn complications, such as acute lung injury (ALI) and adult respiratory distress syndrome (ARDS).

Augmented bacterial elimination by Kupffer cells after IL-18 pretreatment via IFN-γ produced from NK cells in burn-injured mice

We recently demonstrated that IL-18 injections following burn restored IFN-γ production and increased mouse survival after bacterial infection. However, it has yet to be fully elucidated how the IL-18 therapy affects the function of phagocytic cells. We investigated the effect of IL-18 therapy on function and interaction of Kupffer cells and NK cells in burned mice. C57BL/6 mice received a 20% full-thickness burn, followed by multiple injections with IL-18. Although burn-injured mice had decreased expression of IL-18 receptors on the NK/NKT cells 5 days after injury, multiple IL-18 injections restored this expression. IL-18 treatment also augmented Kupffer cell phagocytosis. Although burn decreased the number of CD68 + Kupffer cells with phagocytic activity, IL-18 treatment partially restored their proportion, and augmented phagocytosis-induced ROS production in CD68 + Kupffer cells after the injection of heat-killed Escherichia coli. Consistently, IL-18 restored the impaired E. coli killing activity of Kupffer cells of burn-injured mice. Such Kupffer cell activation by IL-18 was abrogated by the deletion of NK cells or IFN-γ. In conclusion, IL-18 therapy in burn-injured mice enhanced function of CD68 + Kupffer cells via the activation of liver NK cells and augmentation of their IFN-γ production, thereby improving survival after E. coli infection.

Astragalus Polysaccharides Attenuate Postburn Sepsis via Inhibiting Negative Immunoregulation of CD4+CD25high T Cells

BackgroudAstragalus polysaccharides (APS) isolated from one of the Chinese herbs, Astragalus mongholicus, are known to have a variety of immunomodulatory activities. However, it is not yet clear whether APS can exert an effect on the immune functions of regulatory T cells (Tregs). This study was carried out to investigate the effect of APS on the immune function of peripheral blood Tregs in postburn sepsis.Methodology/Principal FindingsBalB/c mice were randomly divided into six groups as follows: sham burn group, burn control(burn without infection animals) group, burn plus P. aeruginosa group, burn plus P. aeruginosa with APS (50 mg/kg) treatment group, burn plus P. aeruginosa with APS (100 mg/kg) treatment group, and burn plus P. aeruginosa with APS (200 mg/kg) treatment group, and they were sacrificed on postburn day 1, 3, 5, and 7, respectively, with seven animals at each time point. Magnetic microbeads were used to isolate peripheral blood Tregs and CD4+ T cells. Phenotypes were analyzed by flow cytometry, and cytokine levels were determined with ELISA. In the burn plus P. aeruginosa group, forkhead/winged helix transcription factor p3 (Foxp3) expression on CD4+CD25+Tregs were strongly enhanced in comparison to the sham group, and the capacity of CD4+CD25+Tregs to produce interleukin (IL)-10 was markedly increased. Administration of APS to inhibit CD4+CD25+Tregs could significantly decrease expression of Foxp3 on CD4+CD25+Tregs, and IL-10 production in burned mice with P. aeruginosa infection. At the same time, proliferative activity and expression of IL-2 and IL-2Rα on CD4+ T cells were restored. In contrast, anti-Toll-like receptor 4 (TLR4) antibody could block the effect of APS on Tregs immune function.ConclusionAPS might suppress CD4+CD25+Treg activity, at least in part, via binding TLR4 on Tregs and trigger a shift of Th2 to Th1 with activation of CD4+ T cells in burned mice with P. aeruginosa infection.

Control of MRSA sepsis by CCL1 antisense ODN in severely burned mice (106.23)

Abstract Severely burned mice are very susceptible to MRSA infection. M1Mϕ, a major effector cell in host antibacterial innate immunity, were not generated in mice 1 to 3 weeks after severe burn injury, because these mice carried M2bMϕ predominantly. Previously, CCL1 was shown to be needed to maintain M2bMϕ properties, and the elimination of M2bMϕ was demonstrated in burned mice treated with CCL1 antisense ODN. Therefore, in this study, we tried to control MRSA infection by CCL1 antisense ODN treatment in mice 1 to 3 weeks after burn injury. Burned mice (25% TBSA, 3rd degree flame burn) were treated s.c. with 10 μg/mouse of CCL1 antisense ODN twice a day for 2 days beginning 13 days after burn injury. Fourteen days after burn injury, these mice were infected i.v. with 107 CFU/mouse of MRSA. The severities of MRSA infection were evaluated by (i) the growth of the pathogen in organs and blood and (ii) their mortality rates, as compared with controls (burned mice treated with scrambled ODN). In the results, all burned mice treated with CCL1 antisense ODN survived after MRSA infection, while 83% of burned mice treated with scrambled ODN died. After infection, MRSA grew in the kidneys and blood of burned mice treated with scrambled ODN, while greatly diminished growth of the pathogen was shown in these organs of burned mice treated with CCL1 antisense ODN. These results indicate that MRSA sepsis is effectively controlled in burned mice subjected to CCL1 gene therapy.