Weihua Cui

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.

Endotoxin Priming Improves Clearance of Pseudomonas aeruginosa in Wild-Type and Interleukin-10 Knockout Mice

ABSTRACT Endotoxin (lipopolysaccharide [LPS]) tolerance is an altered state of immunity caused by prior exposure to LPS, in which production of many cytokines, including gamma interferon (IFN-γ) and interleukin-12 (IL-12), are reduced but secretion of the anti-inflammatory cytokine IL-10 is increased in response to a subsequent LPS challenge. This pattern of cytokine production is also characteristic of postinflammatory immunosuppression. Therefore, we hypothesized that LPS-primed mice would exhibit an impaired ability to respond to systemic infection with the opportunistic pathogen Pseudomonas aeruginosa. We further hypothesized that depletion of IL-10 would reverse the endotoxin-tolerant state. To test this hypothesis, systemic clearance of Pseudomonas aeruginosa was measured for LPS-primed wild-type and IL-10-deficient mice. LPS-primed wild-type mice exhibited significant suppression of LPS-induced IFN-γ and IL-12 but increased IL-10 production in blood and spleen compared to levels exhibited by saline-primed wild-type mice. The suppressed production of IFN-γ and IL-12 caused by LPS priming was ablated in the spleens, but not blood, of IL-10 knockout mice. LPS-primed wild-type mice cleared Pseudomonas aeruginosa from lungs and blood more effectively than saline-primed mice. LPS-primed IL-10-deficient mice were particularly efficient in clearing Pseudomonas aeruginosa after systemic challenge. These studies show that induction of LPS tolerance enhanced systemic clearance of Pseudomonas aeruginosa and that this effect was augmented by neutralization of IL-10.

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.

Dendritic Cell Modification of Neutrophil Responses to Infection after Burn Injury

Burn patients are highly susceptible to infections due to increased exposure through wounds and impairments in a number of immune functions. Dendritic cells (DCs) are important in activation of numerous immune responses that are essential for the clearance of infections. We have found that prophylactic treatment of burn-injured mice with the DC growth factor FLT3 ligand (FL) significantly increases resistance to burn wound infections in a DC-dependent manner that is correlated closely with enhanced bacterial clearance. However, as DCs are not typically microbicidal, the mechanisms by which DC modulation enhances bacterial clearance are not known. Due to the rapid response of neutrophils to cutaneous wounds, and the reported interactions between DCs and neutrophils, we investigated the role of neutrophils in FL-mediated resistance to burn wound infection. This was examined both in vivo and in vitro through neutrophil depletion, supplementation of neutrophils, and assessment of neutrophil chemotaxis following FL treatment. To test the involvement of DCs, CD11c-diphtheria toxin receptor transgenic mice were used to deplete DCs during FL treatment. Studies revealed that neutrophils do play a critical role in FL-mediated resistance to a burn wound infection. Additionally, treatment with FL after a burn injury enhances neutrophil-mediated control of bacterial spread, neutrophil migratory capacity, and myeloperoxidase production in a DC-dependent manner. The results of this study provide new insight into immunological mechanisms that can offer protection against infection after burn injury.

Glucan phosphate treatment attenuates burn-induced inflammation and improves resistance to Pseudomonas aeruginosa burn wound infection.

These studies evaluated the effects treatment with glucan phosphate, a soluble polysaccharide immunomodulator, on the inflammatory response induced by burn injury and on resistance to Pseudomonas aeruginosa burn wound infection. Mice were exposed to 35% total body surface area burns and were resuscitated with lactated Ringers (LR) solution alone or LR supplemented with glucan phosphate (40 mg/kg). Glucan phosphate treatment attenuated burn-induced expression of interleukin (IL)-1β, IL-6, and IL-10 mRNAs in spleen, lung, and heart. Plasma concentrations of IL-1β, IL-6, macrophage inflammatory protein (MIP)-2, and IL-10 were also decreased in burned mice treated with glucan phosphate compared with vehicle-treated controls. Early postburn mortality was not significantly different between control (20%) and glucan phosphate-treated (10%) mice, but there was a small improvement in acid-base balance in the glucan phosphate-treated group. Mice received a second injection of glucan phosphate or LR on day 4 postburn and were infected by topical application of P. aeruginosa to the burn wound on day 5. Glucan phosphate treatment significantly improved survival in mice exposed to P. aeruginosa burn wound infection. The improved survival correlated with lower bacterial burden in the burn wound, attenuated production of proinflammatory cytokines, and enhanced production of Th1 cytokines. These studies show that glucan phosphate treatment attenuates burn-induced inflammation and increases resistance to P. aeruginosa burn wound infection in an experimental model of burn injury.

Insulin increases resistance to burn wound infection-associated sepsis.

Objective:This study was designed to determine the ability of insulin to improve outcome following a Pseudomonas aeruginosa wound infection in a rodent model of severe burn injury. Background:Severe burn injury predisposes patients to burn wound infections that can disseminate, lead to uncontrolled inflammation, and induce septic shock. Whereas insulin administration has been extensively discussed to improve morbidity and mortality in critically ill patients, the ability of insulin to improve outcomes of severely burned patients with infected burn wounds is not known. Design:Sprague-Dawley rats. Setting:University setting. Intervention:Burn-injured Sprague Dawley rats were randomized into treatment groups that received either saline or insulin. Burn wounds were topically inoculated with a lethal dose of Pseudomonas aeruginosa 6 days after injury. Measurements and Main Results:Survival, systemic dissemination of bacteria, systemic inflammation, and immune activation were examined. Insulin decreased the early inflammatory response to a severe burn injury. Treatment with low doses of insulin following burn injury improved the outcome of rats in response to a lethal burn wound infection. Specifically, survival was improved and systemic dissemination of bacteria from the wound was decreased. Systemic inflammation, indicated by serum interleukin-6 levels, was significantly decreased by insulin treatments after injury. Additionally, insulin treatments were associated with alterations in B and T lymphocyte responses to wound infection. Conclusions:Although the mechanisms by which insulin improves outcome following a lethal burn wound infection are not known, the data suggest that immunologic responses to infection may be altered by postburn insulin treatments.

Dendritic cells modulate burn wound healing by enhancing early proliferation

Adequate wound healing is vital for burn patients to reduce the risk of infections and prolonged hospitalization. Dendritic cells (DCs) are antigen presenting cells that release cytokines and are central for the activation of innate and acquired immune responses. Studies have showed their presence in human burn wounds; however, their role in burn wound healing remains to be determined. This study investigated the role of DCs in modulating healing responses within the burn wound. A murine model of full‐thickness contact burns was used to study wound healing in the absence of DCs (CD11c promoter‐driven diphtheria toxin receptor transgenic mice) and in a DC‐rich environment (using fms‐like tyrosine kinase‐3 ligand, FL‐ a DC growth factor). Wound closure was significantly delayed in DC‐deficient mice and was associated with significant suppression of early cellular proliferation, granulation tissue formation, wound levels of TGFβ1 and formation of CD31+ vessels in healing wounds. In contrast, DC enhancement significantly accelerated early wound closure, associated with increased and accelerated cellular proliferation, granulation tissue formation, and increased TGFβ1 levels and CD31+ vessels in healing wounds. We conclude that DCs play an important role in the acceleration of early wound healing events, likely by secreting factors that trigger the proliferation of cells that mediate wound healing. Therefore, pharmacological enhancement of DCs may provide a therapeutic intervention to facilitate healing of burn wounds.

Fms-like tyrosine kinase-3 ligand alters antigen-specific responses to infections after severe burn injury

Burn patients are susceptible to opportunistic infections partly because of decreased immune functions, especially TH1-driven antigen-specific responses, which are regulated by dendritic cells. The dendritic cell growth factor, fms-like tyrosine kinase-3 ligand (FL), has been shown to increase resistance to Pseudomonas aeruginosa, in a dendritic cell-dependent manner, in a mouse model of burn wound infection. The specific mechanisms of protection are not known. This study tested the hypothesis that FL can enhance production of P. aeruginosa-specific antibodies after burn wound infection. Mice that had been previously exposed to P. aeruginosa were infected after burn injury by wound inoculation or challenged by intraperitoneal injection of heat-killed P. aeruginosa. In response to wound infection, FL treatments enhanced bacterial clearance and induced a shift from immunoglobulin (Ig) M toward IgG and IgA. However, serum levels of neither P. aeruginosa-specific antibodies nor interferon &ggr; (IFN-&ggr;) were significantly increased by FL, possibly because of decreased systemic exposure to bacteria. After challenge with heat-killed bacteria, which ensured equal exposures, FL-treated mice produced significantly greater levels of P. aeruginosa-specific IgG2a, which correlated with an increase in serum levels of interferon &ggr; and enhanced opsonization capacity. IL-12, IL-10, and transforming growth factor &bgr; were significantly increased in FL-treated mice, regardless of the type of challenge. These findings indicate that FL treatments after burn injury enhance cytokine responses to recall antigens and increase bacterial clearance. In addition, through its ability to promote TH1-associated antigen-specific responses, FL may have potential as an immunotherapy to enhance adaptive immunity after severe burn injury.ABBREVIATIONS-FL-fms-like tyrosine kinase-3 ligand; HKPA-heat-killed Pseudomonas aeruginosa; LR-lactated Ringers solution

Endogenous Fms-like Tyrosine Kinase-3 Ligand levels are not altered in mice after a severe burn and infection

BackgroundFms-like tyrosine kinase-3 ligand (Flt3L) is a hemopoietic cytokine and dendritic cell (DC) growth factor that promotes the proliferation and differentiation of progenitor cells into DCs. We have previously found that treatment of severely burned mice with recombinant Flt3L significantly enhances DC production and bacterial clearance from infected burn wounds, and increases global immune cell activation and survival in response to a burn wound infection. These significant benefits of Flt3L treatment after burn injury have prompted the question of whether or not severe burn injury induces deficits in endogenous Flt3L levels that could affect DCs and subsequent responses to infection.ResultsTo address this, male BALB/c mice received a 30% total body surface area scald burn. Blood, spleens, and wound-draining lymph nodes were harvested at various time-points after injury. Some mice received a wound inoculation with P. aeruginosa. Murine Flt3L and G-CSF levels were measured by ELISA. Burn injury had no significant effect on Flt3L levels at any post-burn time-point examined compared to normal Flt3L levels in the sera, spleen, or lymph nodes. Additionally, Flt3L levels in the sera, spleen, and lymph nodes were not significantly altered when wounds were inoculated on the day of burn injury or at post-burn time points examined. Alternatively, levels of G-CSF were increased in response to burn injury and burn wound infection. Additionally, DC numbers and functions were not altered following burn injury alone. There was no significant difference between the number of DCs in the spleens of sham-injured mice and mice at 5 days after burn injury. When naïve T cells from sham-injured mice were co-cultured with DCs from either sham- or burn-injured mice, IFN-γ production was similar, however, IFN-γ levels produced by T cells harvested from burn-injured mice were significantly lower than those produced by T cells from sham mice, regardless of which DC group, sham or burn, was used in the coculture.ConclusionThese data suggest that the beneficial effects of Flt3L treatments after burn injury are not due to correction of a burn-associated Flt3L deficiency but rather, are likely due to supplementary stimulation of DC production and immune responses to infection.

Fms-Like Tyrosine Kinase-3 Ligand Attenuates Local and Systemic Infection in a Model of Post-Burn Pneumonia

Background: Burn injury induces immunosuppression and promotes infection with opportunistic pathogens. Pneumonia and sepsis are leading causes of post-burn morbidity and mortality. Fms-like tyrosine kinase-3 ligand (Flt3L) improves local and systemic resistance to P aeruginosa-associated burn wound infection. This study evaluates the effects of post-burn prophylactic Flt3L treatment on local and systemic infection and inflammation in a murine model of pneumonia and sepsis. Methods: Mice received a severe scald burn, were treated with Flt3L or vehicle (CTR) for 5 days, and inoculated trans-nasally with P aeruginosa. Lung, blood, and spleen were harvested at 24 and 48 h postinoculation (p.i.) to assess infection (bacterial burden, bacteremia, distant organ manifestation) and inflammation (interleukin-6 (IL-6) and myeloperoxidase (MPO) levels). Histology correlated infection and inflammation parameters with morphology. Survival at various bacterial concentrations was monitored for 14 days p.i. Results: Bacterial burden was significantly reduced in lung and spleen of Flt3L-treated mice. Flt3L treatment was associated with decreased signs of pulmonary inflammation (reduced wet weight and IL-6 levels), lower incidences of bacteremia and septic distant organ manifestation, and reduced systemic inflammation (IL-6 and MPO). Histologically, reduced alveolar and peribronchiolar neutrophil and lymphocyte infiltration indicated attenuated pulmonary inflammation after Flt3L treatment. Overall survival was comparable between groups for all doses of P aeruginosa, but mortality delayed in the Flt3L-treated group. Conclusion: Prophylactic treatment with Flt3L could augment antimicrobial therapy of post-burn pneumonia through improvement of the initial host response to challenge with P aeruginosa, attenuate local, and systemic inflammation as well as septic pathogen dissemination.