Prophylactic treatment with fms-like tyrosine kinase-3 ligand after burn injury enhances global immune responses to infection

Severely burned patients are susceptible to infections with opportunistic organisms due to altered immune responses and frequent wound contamination. Immunomodulation to enhance systemic and local responses to wound infections may be protective after burn injury. We previously demonstrated that pretreatments with fms-like tyrosine kinase-3 (Flt3) ligand (Flt3L), a dendritic cell growth factor, increase the resistance of mice to a subsequent burn injury and wound infection by a dendritic cell-dependent mechanism. This study was designed to test the hypothesis that Flt3L administration after burn injury decreases susceptibility to wound infections by enhancing global immune cell activation. Mice were treated with Flt3L after burn injury and examined for survival, wound and systemic bacterial clearance, and immune cell activation after wound inoculation with Pseudomonas aeruginosa. To gain insight into the local effects of Flt3L at the burn wound, localization of Langerhans cells was examined. Mice treated with Flt3L had significantly greater numbers of CD25-expressing T cells and CD69-expressing T and B cells, neutrophils, and macrophages after, but not before, infection. Overall leukocyte apoptosis in response to infection was decreased with Flt3L treatment. Survival and local and systemic bacterial clearance were enhanced by Flt3L. Langerhans cells appeared in the dermis of skin bordering the burn wound, and further increased in response to wound infection. Flt3L augmented the appearance of Langerhans cells in response to both injury and infection. These data suggest that dendritic cell enhancement by Flt3L treatments after burn injury protects against opportunistic infections through promotion of local and systemic immune responses to infection.     

IFN-gamma production from liver mononuclear cells of mice in burn injury as well as in postburn bacterial infection models and the therapeutic effect of IL-18

Hosts after severe burn injury are known to have a defect in the Th1 immune response and are susceptible to bacterial infections. We herein show that liver NK cells are potent IFN-gamma producers early after burn injury. However, when mice were injected with LPS 24 h after burn injury, IFN-gamma production from liver mononuclear cells (MNC; which we previously showed to be NK cells) was suppressed, and the serum IFN-gamma concentration did not increase, while serum IL-10 conversely increased compared with control mice. Interestingly, a single injection of IL-18 simultaneously with LPS greatly restored the serum IFN-gamma concentration in mice with burn injury and also increased IFN-gamma production from liver MNC. Nevertheless, a single IL-18 injection into mice simultaneously with LPS was no longer effective in the restoration of serum IFN-gamma and IFN-gamma production from the liver MNC at 7 days after burn injury, when mice were considered to be the most immunocompromised. However, IL-18 injections into mice on alternate days beginning 1 day after burn injury strongly up-regulated LPS-induced serum IFN-gamma levels and IFN-gamma production from liver and spleen MNC of mice 7 days after burn injury and down-regulated serum IL-10. Furthermore, similar IL-18 therapy up-regulated serum IFN-gamma levels in mice with experimental bacterial peritonitis 7 days after burn injury and greatly decreased mouse mortality. Thus, IL-18 therapy restores the Th1 response and may decrease the susceptibility to bacterial infection in mice with burn injury.     

Gut-associated lymphoid T cell suppression enhances bacterial translocation in alcohol and burn injury

The mechanism of alcohol-mediated increased infection in burn patients remains unknown. With the use of a rat model of acute alcohol and burn injury, the present study ascertained whether acute alcohol exposure before thermal injury enhances gut bacterial translocation. On day 2 postinjury, we found a severalfold increase in gut bacterial translocation in rats receiving both alcohol and burn injury compared with the animals receiving either injury alone. Whereas there were no demonstrable changes in intestinal morphology in any group of animals, a significant increase in intestinal permeability was observed in ethanol- and burn-injured rats compared with the rats receiving either injury alone. We further examined the role of intestinal immune defense by determining the gut-associated lymphoid (Peyer's patches and mesenteric lymph nodes) T cell effector responses 2 days after alcohol and burn injury. Although there was a decrease in the proliferation and interferon-gamma by gut lymphoid T cells after burn injury alone; the suppression was maximum in the group of rats receiving both alcohol and burn injuries. Furthermore, the depletion of CD3(+) cells in healthy rats resulted in bacterial accumulation in mesenteric lymph nodes; such CD3(+) cell depletion in alcohol- and burn-injured rats furthered the spread of bacteria to spleen and circulation. In conclusion, our data suggest that the increased intestinal permeability and a suppression of intestinal immune defense in rats receiving alcohol and burn injury may cause an increase in bacterial translocation and their spread to extraintestinal sites.     

Injury-induced suppression of effector T cell immunity requires CD1d-positive APCs and CD1d-restricted NKT cells

Overwhelming infection remains the leading cause of death from serious burn injury despite recent advances in the care of burn patients and a better understanding of immune and inflammatory consequences of injury. In this study, we report a critical requirement for CD1d-restricted NKT cells and CD1d expression by APCs in the immune dysfunction that occurs early after burn injury. Using a well-established murine scald injury model with BALB/c and BALB/c CD1d knockout mice, we investigated whether peripheral T cell immunity was affected by the presence or absence of CD1d-restricted NKT cells in the early stages after injury. Using Ag-specific delayed-type hypersensitivity, T cell proliferation, and cytokine production as indices of immune responsiveness, we observed that both CD1d expression by APCs and CD1d-restricted NKT cells are required for immune suppression after injury. Via adoptive transfer of splenocytes from injured mice to uninjured recipients, we found injury-induced suppression of immunity to be Ag specific, long lasting, and critically dependent on cell surface expression of CD1d by APCs. Together, our results suggest that the defects in T cell responsiveness that occur subsequent to severe burn injury are not merely the result of global or passive suppression, but instead represent an active form of CD1d/NKT cell-dependent immunologic tolerance.     

Flagellin Treatment Prevents Increased Susceptibility to Systemic Bacterial Infection after Injury by Inhibiting Anti-Inflammatory IL-10+ IL-12- Neutrophil Polarization

Severe trauma renders patients susceptible to infection. In sepsis, defective bacterial clearance has been linked to specific deviations in the innate immune response. We hypothesized that innate immune modulations observed during sepsis also contribute to increased bacterial susceptibility after severe trauma. A well-established murine model of burn injury, used to replicate infection following trauma, showed that wound inoculation with P. aeruginosa quickly spreads systemically. The systemic IL-10/IL-12 axis was skewed after burn injury with infection as indicated by a significant elevation in serum IL-10 and polarization of neutrophils into an anti-inflammatory (“N2”; IL-10⁺ IL-12⁻) phenotype. Infection with an attenuated P. aeruginosa strain (ΔCyaB) was cleared better than the wildtype strain and was associated with an increased pro-inflammatory neutrophil (“N1”; IL-10⁻IL-12⁺) response in burn mice. This suggests that neutrophil polarization influences bacterial clearance after burn injury. Administration of a TLR5 agonist, flagellin, after burn injury restored the neutrophil response towards a N1 phenotype resulting in an increased clearance of wildtype P. aeruginosa after wound inoculation. This study details specific alterations in innate cell populations after burn injury that contribute to increased susceptibility to bacterial infection. In addition, for the first time, it identifies neutrophil polarization as a therapeutic target for the reversal of bacterial susceptibility after injury.     

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.     

Up-regulation of cell surface Toll-like receptors on circulating γδ T-cells following burn injury

Burn injury is associated with profound inflammation and activation of the innate immune system involving γδ T-cells. Similarly, Toll-like receptors (TLR) are associated with activation of the innate immune response; however, it is unclear whether TLR expression is altered in γδ T-cells after major burn injury. To study this, male C57BL/6 mice were subjected to burn injury (25% TBSA) and 1 or 7 days thereafter, blood and spleen cells were isolated and subjected to FACs analysis for TLRs and other phenotypic markers (γδ TCR, αβ TCR, CD69, CD120b). A marked increase in the number of circulating γδ T-cells was observed at 24 h post-burn (14% vs. 4%) and a higher percentage of these cells expressed TLR-2. TLR-4 expression was also increased post-burn, but to a lesser degree. These changes in TLR expression were not associated with altered CD69 or CD120b expression in γδ T-cells. The mobilization of, and increased TLR expression in, γδ T-cells was transient, as phenotypic changes were not evident at 7 days post-burn or in γδ T-cells from the circulation or spleen. The increases in TLR expression were not observed in αβ T-cells after burn injury. In conclusion, 24 h after burn injury mobilization of γδ T-cells with increased TLR expression was observed. This finding suggests that this unique T-cell population is critical in the innate immune response to injury, possibly through the recognition of danger signals by TLRs.     

IL-15 Superagonist Expands mCD8+ T,NK and NKT Cells after Burn Injury but Fails to Improve Outcome during Burn Wound Infection

Background

Severely burned patients are highly susceptible to opportunistic infections and sepsis, owing to the loss of the protective skin barrier and immunological dysfunction. Interleukin-15 (IL-15) belongs to the IL-2 family of common gamma chain cytokines and stimulates the proliferation and activation of T (specifically memory CD8), NK and NKT cells. It has been shown to preserve T cell function and improve survival during cecal ligation and puncture (CLP)-induced sepsis in mice. However, the therapeutic efficacy of IL-15 or IL-15 superagonist (SA) during infection after burn injury has not been evaluated. Moreover, very few, if any, studies have examined, in detail, the effect of burn injury and infection on the adaptive immune system. Thus, we examined the effect of burn and sepsis on adaptive immune cell populations and the effect of IL-15 SA treatment on the host response to infection.

Methods

Mice were subjected to a 35% total body surface area burn, followed by wound infection with Pseudomonas aeruginosa. In some experiments, IL-15 SA was administered after burn injury, but before infection. Leukocytes in spleen, liver and peritoneal cavity were characterized using flow cytometry. Bacterial clearance, organ injury and survival were also assessed.

Results

Burn wound infection led to a significant decline in total white blood cell and lymphocyte counts and induced organ injury and sepsis. Burn injury caused decline in CD4⁺ and CD8⁺ T cells in the spleen, which was worsened by infection. IL-15 treatment inhibited this decline and significantly increased cell numbers and activation, as determined by CD69 expression, of CD4⁺, CD8⁺, B, NK and NKT cells in the spleen and liver after burn injury. However, IL-15 SA treatment failed to prevent burn wound sepsis-induced loss of CD4⁺, CD8⁺, B, NK and NKT cells and failed to improve bacterial clearance and survival.

Conclusion

Cutaneous burn injury and infection cause significant adaptive immune dysfunction. IL-15 SA does not augment host resistance to burn wound sepsis in mice despite inducing proliferation and activation of lymphocyte subsets.     

Relationships between burn size,immunosuppression, and macrophage hyperactivity in a murine model of thermal injury

Burn injury induces immune dysfunction and alters numerous physiological parameters. While clinical studies indicate that burn injury size profoundly impacts patient immune status, only limited experimental studies have systematically addressed its impact on immune functional parameters. In the present study, mice were subjected to burn injuries of varying sizes and splenic immune cells (splenocytes and macrophages) were isolated 7 days thereafter. Burn injury suppressed splenic T-cell proliferation in an injury size-dependent manner that correlated with the release of the immunosuppressive mediators PGE(2) and nitric oxide. In addition, a shift towards an immunosuppressive Th-2 cytokine profile and a hyperactive macrophage phenotype (increased release of inflammatory mediators) was observed post-injury, however, this effect was in part independent of burn size. Thus, unlike patient survival data, burn injury-induced changes in immune function do not necessarily correlate with the size of the injury.     

Burn-induced increase in atrogin-1 and MuRF-1 in skeletal muscle is glucocorticoid independent but downregulated by IGF-I

The present study determined whether thermal injury increases the expression of the ubiquitin (Ub) E3 ligases referred to as muscle ring finger (MuRF)-1 and muscle atrophy F-box (MAFbx; aka atrogin-1), which are muscle specific and responsible for the increased protein breakdown observed in other catabolic conditions. After 48 h of burn injury (40% total body surface area full-thickness scald burn) gastrocnemius weight was reduced, and this change was associated with an increased mRNA abundance for atrogin-1 and MuRF-1 (3.1- to 8-fold, respectively). Similarly, burn increased polyUb mRNA content in the gastrocnemius twofold. In contrast, there was no burn-induced atrophy of the soleus and no significant change in atrogin-1, MuRF-1, or polyUb mRNA. Burns also did not alter E3 ligase expression in heart. Four hours after administration of the anabolic agent insulin-like growth factor (IGF)-I to burned rats, the mRNA content of atrogin-1 and polyUb in gastrocnemius had returned to control values and the elevation in MuRF-1 was reduced 50%. In contrast, leucine did not alter E3 ligase expression. In a separate study, in vivo administration of the proteasome inhibitor Velcade prevented burn-induced loss of muscle mass determined at 48 h. Finally, administration of the glucocorticoid receptor antagonist RU-486 did not prevent burn-induced atrophy of the gastrocnemius or the associated elevation in atrogin-1, MuRF-1, or polyUb. In summary, the acute muscle wasting accompanying thermal injury is associated with a glucocorticoid-independent increase in the expression of several Ub E3 ligases that can be downregulated by IGF-I.     

Thioredoxin-interacting protein mediates NALP3 inflammasome activation in podocytes during diabetic nephropathy

Numerous studies have shown that the NALP3 inflammasome plays an important role in various immune and inflammatory diseases. However, whether the NALP3 inflammasome is involved in the pathogenesis of diabetic nephropathy (DN) is unclear. In our study, we confirmed that high glucose (HG) concentrations induced NALP3 inflammasome activation both in vivo and in vitro. Blocking NALP3 inflammasome activation by NALP3/ASC shRNA and caspase-1 inhibition prevented IL-1β production and eventually attenuated podocyte and glomerular injury under HG conditions. We also found that thioredoxin (TRX)-interacting protein (TXNIP), which is a pro-oxidative stress and pro-inflammatory factor, activated NALP3 inflammasome by interacting with NALP3 in HG-exposed podocytes. Knocking down TXNIP impeded NALP3 inflammasome activation and alleviated podocyte injury caused by HG. In summary, the NALP3 inflammasome mediates podocyte and glomerular injury in DN, moreover, TXNIP participates in the formation and activation of the NALP3 inflammasome in podocytes during DN, which represents a novel mechanism of podocyte and glomerular injury under diabetic conditions.     

The Pseudomonas aeruginosa toxin L-2-amino-4-methoxy-trans-3-butenoic acid inhibits growth and induces encystment in Acanthamoeba castellanii

L-2-Amino-4-methoxy-trans-3-butenoic acid (AMB) is a toxic antimetabolite produced by the opportunistic pathogen Pseudomonas aeruginosa. To evaluate its importance as a potential virulence factor, we tested the host response towards AMB using an Acanthamoeba castellanii cell model. We found that AMB (at concentrations ≥ 0.5 mM) caused amoebal encystment in salt buffer, while inhibiting amoebal growth in rich medium in a dose-dependent manner. However, no difference in amoebal plaque formation was observed on bacterial lawns of wild type and AMB-negative P. aeruginosa strains. We thereby conclude that AMB may eventually act as a virulence factor, but only at relatively high concentrations.     

Enhancement of dendritic cell production by fms-like tyrosine kinase-3 ligand increases the resistance of mice to a burn wound infection

Fms-like tyrosine kinase-3 ligand (Flt3L) is a hemopoietic cytokine that stimulates the production of dendritic cells. This study evaluated the ability of Flt3L-enhanced dendritic cell production to increase the resistance of mice to a burn wound infection with Pseudomonas aeruginosa, a common source of infections in burn patients that have impaired immunity and are susceptible to opportunistic microorganisms. Treatment of mice with Flt3L for 5 days caused a significant increase in dendritic cell numbers in the spleen and significantly increased survival upon a subsequent burn wound infection. Improved survival in Flt3L-treated mice was associated with limited bacterial growth and spread within the burn wounds and a decrease in systemic dissemination of P. aeruginosa. Resistance to burn wound infection could also be conferred to recipient mice by the adoptive transfer of dendritic cells that had been isolated from spleens of Flt3L-treated mice. Adoptive transfer of the same number of splenic dendritic cells from nontreated mice did not confer resistance to burn wound infection. These data indicate that Flt3L can increase the resistance of mice to a P. aeruginosa burn wound infection through both stimulation of dendritic cell production and enhancement of dendritic cell function.     

Burn injury initiates a shift in superantigen-induced T cell responses and host survival

Severe injury induces a temporal shift in immune reactivity that can cause serious complications or even death. We previously reported that mice exposed to bacterial superantigen (SAg) early after injury undergo a strong SAg response with lethal consequences. This study compares the early and late effects of burn injury on SAg reactivity in vivo to establish how injury influences adaptive immune responses. We found that mice challenged with ordinarily sublethal doses of staphylococcal enterotoxin A or staphylococcal enterotoxin B at 1 day after burn injury exhibited high mortality, whereas no mortality occurred at 7 days after injury. This shift in mortality correlated with higher Th2-type cytokines (IL-4 and IL-10) being expressed by CD4(+) and CD8(+) T cells from burn as opposed to sham mice at 7 days after injury. Lymph node cells from burn-injured mice also produced higher levels of Th2-type cytokines at 7 days after injury. The results of cell-mixing studies using CD4(+) and CD8(+) T cells mixed with APCs from sham or burn mice suggested that changes in both T cells and APCs are involved in the altered SAg response. Finally, the biological significance of altered SAg reactivity following injury was shown by demonstrating that blocking IL-10 activity in vivo caused higher SAg-induced mortality at 7 days after injury. These findings support the idea that injury promotes a Th2-type shift in adaptive immune reactivity. Although prior studies link this counterinflammatory-type response to lowered resistance to infection, the present results suggest it may sometimes benefit the injured host.     

Effects of insulin-like growth factor-1/binding protein-3 complex on muscle atrophy in rats

Muscle atrophy and wasting is a serious problem that occurs in patients with prolonged debilitating illness, burn injury, spinal injury, as well as with space flight. Current treatment for such atrophy, which often relies on nutritional supplementation and physical therapy, is of limited value in preventing the muscle wasting that occurs. Considerable recent attention has focused on the use of anabolic growth factors such as insulin-like growth factor (IGF-1) in preventing muscle atrophy during limb disuse or with various catabolic conditions. However, potential side effects such as hypoglycemia appear to be limiting factors in the usefulness of IGF-1 for clinical treatment of muscle wasting conditions. The formulation of IGF-1 used in this study (IGF-1/BP3) is already bound to its endogenous-binding protein (BP3) and, as a result, has a greater specificity of action and significantly less hypoglycemic effect. Using a rat model of hind limb suspension (HLS) for 10 days, we induced marked muscle atrophy that was accompanied by enhanced muscle proteolysis and reduced muscle protein content. When HLS rats were treated with IGF-1/BP3 (50 mg/kg, b.i.d.), they retained greater body and muscle mass. Muscle protein degradation was significantly reduced and muscle protein content was preserved. The rate of protein synthesis, although somewhat reduced in HLS muscle, was not significantly elevated by IGF-1/BP3 treatment. Volume density of HLS-treated muscles were increased compared to untreated HLS rats and the actual number of fibers per area of muscle was likewise increased. The results of the current study suggest that IGF-1/BP3 might be useful for inhibiting muscle proteolysis in catabolic conditions and thus preserving muscle protein content and mass.     

Nitric oxide contributes to the development of a post-injury Th2 T-cell phenotype and immune dysfunction

Severe injury induces immune dysfunction resulting in increased susceptibility to opportunistic infections. Previous studies from our laboratory have demonstrated that post-burn immunosuppression is mediated by nitric oxide (NO) due to the increased expression of macrophage inducible nitric oxide synthase (iNOS). In contrast, others suggest that injury causes a phenotypic imbalance in the regulation of Th1- and Th2 immune responses. It is unclear whether or not these apparently divergent mediators of immunosuppression are interrelated. To study this, C57BL/6 mice were subjected to major burn injury and splenocytes were isolated 7 days later and stimulated with antiCD3. Burn injury induced NO-mediated suppression of proliferative responses that was reversed in the presence of the NOS inhibitor L-monomethyl-L-arginine and subsequently mimicked by the addition of the NO donor, S-nitroso-N-acetyl-penicillamine (SNAP). SNAP also dose-dependently suppressed IFN-gamma and IL-2 (Th1), but not IL-4 and IL-10 (Th2) production. Delaying the addition of SNAP to the cultures by 24 h prevented the suppression of IFN-gamma production. The Th2 shift in immune phenotype was independent of cGMP and apoptosis. The addition of SNAP to cell cultures also induced apoptosis, attenuated mitochondrial oxidative metabolism and induced mitochondrial membrane depolarization. However, these detrimental cellular effects of NO were observed only at supra-physiologic concentrations (>250 microM). In conclusion, these findings support the concept that NO induces suppression of cell-mediated immune responses by selective action on Th1 T cells, thereby promoting a Th2 response.     

Burn injury-induced alterations in wound inflammation and healing are associated with suppressed hypoxia inducible factor-1alpha expression

A major complication associated with burn injury is delayed wound healing. While healing of the burn injury site is essential, healing of distal injury sites caused by surgical interventions and other processes also is important. The impact of burn injury on healing of these distal wound sites is not understood clearly. To study this, mice were subjected to major burn injury or a sham procedure. Immediately following, excisional wounds were made on the dorsal surface caudal to the burn site and wound closure was monitored over a 7-d period by planimetry. In a second series of experiments, plasma and excisional wounds were collected for in vitro analysis of cyto- and chemokine levels, L-arginine metabolism, and hypoxia-inducible factor (HIF)-1alpha expression. At 1-7 d post-injury, a significant inflammatory response was evident in both groups, but the healing process was delayed in the burn-injured mice. At 3 d post-injury, wound levels of tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and keratinocyte-derived chemokine were suppressed in the burn group. This difference in the wound inflammatory response was independent of changes in L-arginine metabolism (nitrate levels, inducible nitric oxide synthase expression, arginase activity), but correlated with a marked reduction in HIF-1alpha protein levels. In conclusion, these findings suggest that HIF-1alpha and the inflammatory response play a significant role in wound healing, and reduced levels of HIF-1alpha contribute to the impaired healing response post-burn.