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The Journal of Immunology, 2008, 180, 3038 -3048
Copyright © 2008 by The American Association of Immunologists, Inc.
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Prophylactic Treatment with Fms-Like Tyrosine Kinase-3 Ligand after Burn Injury Enhances Global Immune Responses to Infection1

Julia Bohannon{dagger}, Weihua Cui*, Robert Cox*,{ddagger}, Rene Przkora{ddagger}, Edward Sherwood*,{ddagger} and Tracy Toliver-Kinsky2,*,{ddagger}

* Department of Anesthesiology, {dagger} Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX 77555, and {ddagger} Shriners Hospitals for Children, Galveston Burn Unit, Galveston, TX 77550


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
For severely burned patients, the significant risk of exposure to opportunistic organisms through open wounds combined with often inadequate immune responses upon exposure, and the emergence of antibiotic-resistant bacterial strains, necessitates the development of treatments to prophylactically enhance the immune system to prevent infections after burn injury. An obstacle to the development of effective immunomodulatory agents is the large number of alterations that has been reported as a consequence of burn injury. Burn-induced alterations in both innate and acquired immunities have been reported, associated with altered macrophage (1, 2), neutrophil (3), NK cell (4, 5), and T cell functions. Cytokine responses to immunological stimuli after burn injury are often altered, including decreases in Th1, relative to Th2, cytokine production (6, 7, 8) and increased production of proinflammatory cytokines (9). An incomplete understanding of the contributions of each of these alterations to protective and deleterious responses to infection after burn injury further complicates the development of specific immunomodulatory agents. Therefore, prophylactic therapies designed to "prime" the immune system for a globally enhanced response in the case of an infection may be beneficial.

Fms-like tyrosine kinase-3 (Flt3)3 ligand (Flt3L) is an agent that has been examined as an immunological adjuvant to enhance global responses to vaccinations (10, 11), cancer cells (12), and infectious organisms (13, 14, 15). Flt3L is a dendritic cell (DC) growth factor that drives the production of DC from progenitor cells of myeloid and lymphoid lineages (16). DC play a central role in the recognition of infection and subsequent activation of innate and acquired immune responses. Upon recognition of microorganisms, DC produce macrophage-, NK cell- and neutrophil-activating cytokines (17, 18, 19, 20), migrate to lymph nodes, and present Ag and costimulatory signals to T cells to activate adaptive immunity (21, 22). Therefore, expansion of DC should affect the activation of multiple immune responses. Indeed, in vivo expansion of DC by Flt3L treatments promotes the induction of {gamma}-IFN, IL-12, and Th-1 responses, the expansion of Ag-specific T cells, and Ag-specific Ab production (23, 24, 25). Flt3L also overcomes conditions of immunological tolerance and suppression associated with inadequate vaccination and endotoxin tolerance, respectively (26, 27).

We previously reported that Flt3L pretreatments, given before burn and infection, increase resistance to Pseudomonas aeruginosa in a mouse model of burn wound infection. Resistance could be conferred by DC from Flt3L-treated mice (28), leading to the hypothesis that Flt3L indirectly increases resistance to infection by enhancing DC interactions with, and activation of, other effector cells of the immune response. This study was designed to test the hypothesis that expansion or functional enhancement of DC by Flt3L treatments leads to enhancement of global immune responses upon infection. Flt3L treatments were administered after burn injury as a prophylactic agent to decrease susceptibility to a burn wound infection.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

All animal procedures were consistent with the National Institutes of Health guidelines for the care and use of experimental animals and were approved by the Institutional Animal Care and Use Committee at the University of Texas Medical Branch. A widely used technique for induction of full-thickness scald burns was used as previously described (29, 30, 31). Briefly, male BALB/c mice, 6–8 wk of age, were anesthetized with 2.5% isoflurane, and the dorsal and lateral surfaces were shaved with clippers. Mice were placed on their backs and secured in a protective template with an opening corresponding to 30% of the total body surface area, and the exposed skin was immersed in 97°C water for 10 s. Lactated Ringers solution (2 ml) was administered i.p. for fluid resuscitation and buprenorphine (2 mg/kg) was given for analgesia. Sham-injured mice were subjected to all of the procedures except immersion in water. Flt3L (10 µg in 0.1 ml of Lactated Ringers solution) treatments were administered by i.p. injection once daily. Control treatment mice received injections of Lactated Ringers solution alone. Recombinant human Flt3L was provided by Amgen. Unless otherwise indicated, treatments were started immediately after burn injury.

Inoculation

P. aeruginosa was used because it is a common source of wound infections and pneumonia in burn patients (32, 33, 34, 35). P. aeruginosa was purchased from the American Type Culture Collection (catalog no. 19660) and is the same strain used by others investigating P. aeruginosa burn wound infections (30). Cultures were grown in tryptic soy broth and diluted in sterile saline solution for wound inoculation. Three days after burn injury, 8000 CFU P. aeruginosa were applied to the surface of the wound. To examine bacterial growth or clearance, inoculated sections of burn wounds were aseptically harvested 72 h after inoculation and were weighed, minced, and homogenized in sterile saline solution. Serial dilutions of tissue homogenates were grown overnight on tryptic soy agar for determination of CFU per gram tissue wet weight. For survival studies, mice were monitored daily for up to 3 wk following wound inoculation. For studies examining systemic clearance, 5 x 107 CFU P. aeruginosa were administered by i.p. injection. To mimic the time frame of systemic dissemination of bacteria from the burn wound, which becomes detectable 3–4 days following wound inoculation, i.p. injections of P. aeruginosa were given 7 days following burn injury. Blood and tissues were harvested 16 h later and cultured overnight.

FACS analysis

Spleens and wound draining lymph nodes (axillary, inguinal) were harvested 72 h after wound inoculation. Tissues were harvested 72 h after inoculation because this duration is the time at which mortalities typically begin to occur. Axillary and inguinal nodes were confirmed as wound-draining in earlier experiments in which dye was injected into the burn wound (data not shown). The spleen and lymph nodes from individual mice were pooled for preparation of single cell suspensions as previously described (36). Leukocytes (106) were incubated with 0.5 µg of Abs of interest for 30 min at 4°C, washed in PBS, and collected by centrifugation at 300 x g for 5 min at 4°C. Cells were reconstituted in 250 µl of 1% paraformaldehyde and analyzed with a FACScan flow cytometer (BD Biosciences). Specific staining was determined by comparison with appropriate Ab isotype controls. Fluorescence-conjugated Abs were purchased from Caltag Laboratories.

Apoptosis was analyzed using a Vybrant Apoptosis Assay kit from Invitrogen Life Technologies according to manufacturer’s recommendations. Briefly, cells were incubated with Annexin V-FITC and propidium iodide for 15 min at room temperature before analysis by flow cytometry. For examination of apoptosis in specific cells types, cells were incubated with cell surface-specific Abs as described, washed, and incubated with Annexin V-FITC for 15 min before analysis.

ELISA

Sera were harvested from mice 72 h after wound inoculation for IL-6 measurements by ELISA (eBioscience). Briefly, standards and samples were incubated for 2 h in microtiter wells that were coated with IL-6 capture Abs, then washed before incubation (2 h) with HRP-conjugated Abs to IL-6. Wells were washed, substrate solution was added, and the reactions were terminated by addition of stop solution. Levels of IL-6 were determined by comparison of absorbance at 450 nm with standards of known concentration.

Immunohistochemistry

Sections of burn wounds and adjacent, nonburned skin were harvested and fixed in Streck Tissue Fixative (Streck Laboratories), paraffin-embedded, and sectioned at 4 µM. For immunolocalization, sections were deparaffinized, and nonspecific labeling was blocked by incubating the sections with normal rabbit serum for 30 min followed by application of the primary Ab CD207 (1/3000; Santa Cruz Biotechnology). Following overnight incubation at 4°C, sections were washed and primary Ab localization accomplished by incubating the sections with biotinylated rabbit anti-goat IgG (1/200; Vector Laboratories) for 30 min, washing and incubation with ABC reagent for 30 min. Visualization of the bound Ab/enzyme complex was accomplished with an alkaline phosphatase detection kit (Vector Laboratories). Sections were then counterstained with hematoxylin. The number of Langerhans cells in the dermal tissues surrounding the burn wounds was measured by a pathologist masked to the specimen status. Each histological sample contained a cross-section of the burned wound with normal tissue on each end. To measure Langerhans cells in the adjacent dermal tissue, the number of positively stained cells was counted in three nonoverlapping, 40x objective fields (at a magnification x400) of view immediately below the epidermis, starting at the wound margin and moving away from the burned area. The measurements from each end of the wounds were combined.

Statistics

Two group comparisons were made with an unpaired, two-tailed Student’s t test, multigroup comparisons were performed with one-way ANOVA and Tukey-Kramer multiple comparison test, correlations were analyzed by a two-tailed Pearson’s test, and survival curves were analyzed by log-rank test, using GraphPad Prism version 4.00 for Windows, GraphPad software. A value of p ≤ 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
DC expansion

In the current study, treatments were given for only four consecutive days, which was sufficient to induce a significant (p < 0.05) increase in the number of CD11c+/MHC class II+ DC in the spleen and skin draining lymph nodes of mice (Fig. 1). DC numbers were similarly and significantly increased in sham and burn-injured mice after Flt3L treatments, compared with Lactated Ringers solution-treated controls. At 72 h after burn wound inoculation, Flt3L-treated mice had an even greater increase in DC numbers. Flt3L-treated mice had significantly more DC in the spleen and skin draining lymph nodes after infection than did the Flt3L-treated burned mice that were not infected (p < 0.01), suggesting enhanced mobilization of DC in response to the infection.


Figure 1
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  		FIGURE 1. Treatment of burned mice with Flt3L for only 4 days increases DC numbers. Sham and burned mice were injected once daily with Flt3L or an equivalent volume of Lactated Ringers solution as a control treatment for 4 days, beginning on the day of burn injury. Three days after burn injury, wounds of mice in the infection group (burn inf) were inoculated with 8000 CFU P. aeruginosa. Spleens and wound draining (axillary, inguinal) lymph nodes were harvested 72 h later, and single cell suspensions were stained with Abs to CD11c and MHC class II (I-Ad) and analyzed by FACS. Sham mice were neither burned nor infected. Burned mice were burned but not infected. Data show the mean number ± SEM of CD11c+/MHC class II+ cells in spleen and lymph node samples. *, p < 0.05, unpaired, two-tailed t test (n = 3 mice/group), significantly different from all other groups; #, p < 0.05, unpaired, two-tailed t test (n = 3 mice/group), significantly different from sham Flt3L and burn Flt3L groups.

 
Immune cell activation after burn wound infection

We examined surface expression of CD69, a commonly used very early activation marker (37, 38) in the spleen and wound draining lymph nodes as an indication of immune cell activation 3 days following wound inoculation. This time point was chosen because this is the time at which mortalities typically begin to occur in nontreated mice. Approximately 21.5% of the leukocytes expressed CD69 in sham mice, and the percentage of CD69+ leukocytes was not significantly increased in response to burn injury or in 4 days of Flt3L treatments (Fig. 2A). Specifically 6 days after burn injury, 24% of leukocytes were CD69+ in burned mice receiving the control Lactated Ringers solution treatment, and 26.5% were CD69+ in burned mice receiving Flt3L treatment. Inoculation of burn wounds did not induce CD69 expression in mice receiving the control Lactated Ringers solution treatment, as only 22% of leukocytes were positive 3 days after inoculation. However, there was a significant, nearly 2-fold, increase in the percentage of CD69+ cells 3 days after inoculation in mice that had been treated with Flt3L for 4 days (45.7%). This response was not due simply to an increase in splenic cellularity because the proportion of cells expressing CD69 was higher. Additionally, because some DC coexpress CD69 (39), infection-associated expression of surface CD69 on non-DC populations was also confirmed after setting analysis gates to exclude CD11c+ cells (data not shown).


Figure 2
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  		FIGURE 2. Flt3L increases the number of CD69-expressing effector cells in response to burn wound infection. Three days after burn injury, mice were inoculated with 8000 CFU P. aeruginosa, as described in Fig. 1. Spleens and wound draining (axillary, inguinal) lymph nodes were harvested 72 h later, and single cell suspensions were stained with Abs to cell surface markers and CD69 and analyzed by FACS. A, Data show the percentage of CD69+ leukocytes as mean ± SEM. *, p ≤ 0.05, significant difference from burn+FL inf group (n = 3–4 mice/group). B, Representative histograms show CD69 after gating on CD3+ T cells (y-axis shows events on scale of 0–16), CD19+ B cells (y-axis scale of 0–16), Ly-6G+ neutrophils (y-axis scale 0–8), F4/80+ macrophages (y-axis scale of 0–24), and DX5+ NK cells (y-axis scale 0–8). C, Data show the mean ± SD of the total number of each cell type, both CD69– (nonactivated) ({square}) and CD69+ (activated) (Figure 2). *, p ≤ 0.05 (n = 6 mice/group), significant difference in the number of CD69+ corresponding cell type by unpaired, two-tailed t test.

 
To identify cells responding to the wound infection, we examined surface expression of CD69 on multiple effector cells. Total CD3+ T cells showed very little expression of CD69 under normal conditions (Fig. 2B, sham) and 6 days after burn injury alone. Interestingly, there was very little change in the number of CD69+ T cells in the spleens and lymph nodes of control-treated (Lactated Ringers solution) mice at 72 h following wound inoculation. However, there was a significant increase in the number of CD3+ cells expressing CD69 after wound inoculation in the Flt3L-treated mice (Fig. 2C). Specifically, the mean number of CD69+CD3+ cells in Flt3L-treated mice was 2.14 times greater than the number in the control-treated mice (3.7 vs 1.7 x 107, respectively, p = 0.04). The number of CD69–CD3+ cells was not increased by Flt3L treatment, indicating a specific increase in the activated population in response to a wound infection. Similarly, the number of CD69+ B cells (p = 0.05), neutrophils (p = 0.001), and macrophages (p = 0.002) was significantly greater in the infected mice that received Flt3L treatment after burn injury, compared with those receiving control treatment (Fig. 2, B and C). Specifically, the number of CD69+CD19+ cells increased almost 2-fold from 8.98 x 106 in the control-treated mice to 1.66 x 107 in the Flt3L-treated mice 72 h after wound inoculation, and the number of CD69+ neutrophils was approximately four times greater in the Flt3L-treated mice (3.07 x 107) compared with the control-treated mice (7.49 x 106). There were approximately three times more CD69+ macrophages in the Flt3L-treated mice compared with the control-treated mice (4.9 x 107 vs 1.59 x 107, respectively). The number of CD69+ DX5+ NK cells was ~2-fold greater in the Flt3L treatment group (1.99 x 107 compared with 9.9 x 106 in control), but the difference was not statistically significant. Additionally, there were no statistically significant differences in the numbers of CD69– T cells, B cells, neutrophils, macrophages, or NK cells. There was, however, a slight (nonsignificant) increase in the number of CD69– CD19+ B cells and DX5+ NK cells.

The expression of another lymphocyte activation marker, CD25, was also examined on T cells. Only a small percentage of CD3+ T cells coexpressed CD25 in the sham (5.3%) and burned (6.4%) control-treated mice (Fig. 3). Flt3L treatments did not affect the percentages of T cells expressing CD25 in the sham (5.5%) or burned (6.2%) mice. However, 72 h after inoculation of burn wounds, the percentages of T cells coexpressing CD25 were increased to 8.4% in the control-treated mice and 10.5% in the Flt3L-treated mice. In the control-treated mice, the percentage of CD25+ cells was significantly greater after infection when compared with both treatment groups of sham mice (p < 0.05), but not when compared with either treatment group of burned mice. The percentage of CD3+ cells expressing CD25 in response to infection was significantly greater in the Flt3L-treated mice compared with all other experimental and treatment groups (p < 0.05). Additionally, the absolute number of CD25+CD3+ cells in wound draining lymph nodes and spleen after wound infection was significantly greater in the Flt3L treatment group compared with all other experimental and treatment groups (p < 0.05) (Fig. 3). The expression of CD25 on CD19+ B cells was also examined, but no significant increases in response to infection or differences between treatment groups were observed (data not shown).


Figure 3
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  		FIGURE 3. Flt3L-treated mice have an increased number of CD25+ T cells after burn wound infection. Three days after burn injury, mice were inoculated with 8000 CFU P. aeruginosa, as described in Fig. 1. Single cell suspensions were stained for CD3 and CD25 and analyzed by flow cytometry. The percentage and number of CD3+ cells coexpressing the CD25 activation marker are shown. *, p < 0.05 (n = 3 mice/group), significantly different from all others; #, p < 0.05 (n = 3 mice/group) significantly different from burn wound infection treated with Lactated Ringers solution group (burn inf LR).

 
Leukocyte death after burn wound infection

Levels of cell death in leukocytes harvested from wound draining lymph nodes and spleens were analyzed by FACS after staining with Annexin V and propidium iodide. There were no significant differences between treatment groups in the percentages of dead/late apoptotic (Annexin V+/propidium iodide+) cells in any of the experimental groups (Fig. 4A). Seventy-two hours after a burn wound infection, the percentages of dead cells increased in both the control- and Flt3L-treated mice and were significantly higher than in the burn Flt3L-treated (p < 0.05), but not other mice. The percentages of early apoptotic (Annexin V+/propidium iodide–) cells also increased in response to burn wound infection. Control-treated mice had a significantly greater percentage of apoptotic leukocytes 72 h after wound inoculation compared with all other experimental and treatment groups (p < 0.05). Although Flt3L-treated mice had a significantly greater percentage of apoptotic cells after wound infection compared with both sham groups and the control-treated burn group, the percentage was significantly lower than in the control-treated group after wound inoculation (p < 0.05). Examination of Annexin V staining in specific cell populations 72 h after burn wound inoculation indicated a trend toward decreased percentages of apoptotic DC and B cells (Fig. 4) in Flt3L-treated, compared with control-treated, mice. Overall, the percentages of Annexin V+ DC and B cells in the spleen and wound draining lymph nodes were high after a wound infection, compared with cells from noninfected mice (data not shown). Surprisingly, there was a significant increase in the percentage of apoptotic T cells in the Flt3L-treated, compared with control-treated, mice after infection (p < 0.05). There were no significant differences between treatment groups in any of the cell types examined in the absence of a wound infection (data not shown).


Figure 4
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  		FIGURE 4. Effects of Flt3L on cell death in response to burn wound infection. Three days after burn injury, mice were inoculated with 8000 CFU P. aeruginosa, as described in Fig. 1. A, Single cell suspensions were stained with Annexin V-FITC and propidium iodide, or cell surface markers followed by Annexin V-FITC. {ddagger}, p < 0.05 (n = 3) significant difference from burn Flt3L group; *, p < 0.05 (n = 3) significant difference from burn infection Flt3L group; #, p < 0.05 (n = 3) significant difference from all others; +, p < 0.05, (n = 3) significant difference from corresponding control Lactated Ringers solution (LR)-treated group. B, Contour plots show representative samples harvested from mice 72 h after burn wound inoculation. Region 1 contains dead or late apoptotic cells (Annexin V+/propidium iodide+) and region 2 contains early apoptotic cells (Annexin V+/propidium iodide–).

 
Systemic IL-6 in response to bacterial challenge

Given the risk of systemic inflammation in burn patients, and the observed increases in activated immune cells responding to burn wound infection in Flt3L-treated mice, we examined circulating IL-6 as a measure of systemic inflammation. Levels of IL-6 in sera harvested from mice 3 days following burn wound infection were measured by ELISA. In sham (noninjured, noninfected) mice, systemic IL-6 levels were not altered by Flt3L injections (Fig. 5A). At six days following burn injury, in the absence of a wound infection, circulating IL-6 levels remained higher than sham levels, but did not differ significantly between treatment groups. Wound infection led to a significant increase in systemic IL-6 in the control-treated (with Lactated Ringers solution) mice (640.4 pg/ml) 3 days after wound inoculation. However, IL-6 levels in the Flt3L-treated mice were not further increased by wound inoculation (151.9 pg/ml). IL-6 levels after infection in the Lactated Ringers solution group were significantly higher (p < 0.05) than IL-6 levels in all other groups.


Figure 5
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  		FIGURE 5. Systemic IL-6 levels in response to infectious and noninfectious P. aeruginosa challenge. Sham or burned mice were injected once daily with Flt3L or an equivalent volume of Lactated Ringers solution (LR) as a control treatment for 4 days, beginning on the day of burn or sham injuries. A, Three days after burn injury, wounds were inoculated with 8000 CFU P. aeruginosa in the infection group (burn+inf). Sera were harvested 72 h later. B, Seven days after sham or burn injury, mice were injected i.p. with HKPA (5 x 107 CFU before heat killing). Challenge was performed at 7 days postburn to model the systemic dissemination of bacteria that typically occurs 3–4 days after wound inoculation. Sera were harvested 16 h later. Data show the mean ± SEM (n = 3 mice per group). *, p < 0.05 significantly different from all other groups, ANOVA, Tukey-Kramer multiple comparisons test.

 
Because decreased systemic IL-6 after wound infection in the Flt3L-treated mice could be attributed to either decreased dissemination of bacteria from the wound or a decreased inflammatory response to the infection, the response to a noninfectious challenge with P. aeruginosa was examined. Sham and burned mice, both Flt3L- and control-treated, received an i.p. injection of heat-killed P. aeruginosa (HKPA) 7 days after sham or burn injury, and systemic IL-6 was measured 16 h later. Mice were challenged 7 days after injury to model systemic dissemination of bacteria that typically occurs 3–4 days after wound inoculation in our model. Levels of IL-6 were low (<10 pg/ml) in both sham and burned mice that were not challenged with HKPA (Fig. 5B). These levels were at least 10-fold lower than systemic levels in the sham and burned mice from the separate experiment shown in Fig. 5A. Although the reason for this difference is not known, it is possible that the mice shown in Fig. 5B had resolved their inflammatory response to the burn injury and manipulations from the 4 days of treatment because these samples were harvested 8 days after sham or burn injury and 4 days after completion of the 4-day treatment protocol. Samples shown in Fig. 5A were harvested 6 days after sham or burn injury and 2 days after completion of the 4-day treatment protocol. Nonetheless, systemic IL-6 was increased in response to HKPA challenge in both the sham and burned mice (Fig. 5B). Surprisingly, HKPA-induced IL-6 in the sham, control-treated mice was approximately twice that of the burned, control-treated mice. IL-6 induction by HKPA in the sham mice was lower after Flt3L treatments (similar to levels in the burned, HKPA-challenged mice). Flt3L did not alter HKPA-induced systemic IL-6 levels in the burned mice.

Resistance to burn wound infections

Because 4 days of Flt3L treatment, beginning on the day of burn injury, were effective at increasing overall immune cell activation in response to an infection, without increasing systemic IL-6, the ability of Flt3L to decrease mortality after a lethal burn wound infection was examined. We also examined the efficacy of Flt3L when treatments were delayed beyond the day of burn injury. Fig. 6 illustrates the various 4-day treatment protocols that were administered. In one group (FL-0), treatment was initiated on the day of burn injury and terminated on the day of wound inoculation. Treatments were delayed by either 1 or 3 days in the other groups. Specifically, the FL-1 mice began treatment 1 day after burn injury and completed treatment 1 day after wound inoculation, whereas the FL-3 mice began treatment 3 days after burn (on the day of inoculation) and received the last treatment 3 days following wound inoculation. Interestingly, survival was similarly increased with all prophylactic treatment protocols, regardless of when treatment was initiated (Fig. 6). Survival in the control treatment (Lactated Ringer solution) group was only 24% 3 wk following wound inoculation, whereas survival was increased to 55% in the FL-0 group and 60% in the FL-1 and FL-3 mice groups. The increase in survival with all three treatments groups was significantly different from the control treatment group (p = 0.014), but not from one another.


Figure 6
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  		FIGURE 6. Prophylactic treatment of burned mice with Flt3L significantly improves survival upon wound infection. Burned mice were injected once daily with 10 µg of Flt3L or an equivalent volume of Lactated Ringers solution (LR) as a control treatment for 4 days (X) as shown in timeline. Treatment was initiated either on the day of burn injury/3 days before wound inoculation (FL-0, n = 31 mice), 1 day after burn/2 days before inoculation (FL-1, n = 15 mice), or 3 days after burn/on the day of inoculation (FL-3, n = 25 mice). Control treatment (Lactated Ringers solution, n = 38 mice) was initiated on the day of burn. Three days after burn injury, wounds were inoculated with 8000 CFU P. aeruginosa, and survival was monitored for 21 days. Data are pooled from multiple experiments. *, p = 0.014, survival was significantly different by log-rank test.

 
When the 4-day treatment protocol was initiated on the day of injury (FL-0), there was a statistically significant decrease in the mean number of bacteria in the burn wound 72 h after inoculation (3.8 x 109 CFU/gram), compared with the control-treated group (9.5 x 109 CFU/gram; p < 0.05) (Fig. 7). However, when treatment was initiated 1 day after injury (FL-1) or on the day of wound inoculation (FL-3), the mean number of bacteria (5.3 and 7.3 x 109 CFU/gram, respectively) was not significantly different from the control-treated group (9.5 x 109 CFU/gram). There was a strong, negative correlation between the number of bacteria in burn wounds 72 h after inoculation and the number of days before wound inoculation that Flt3L treatment was initiated (Pearson correlation R = –0.9886; p = 0.011).


Figure 7
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  		FIGURE 7. Bacterial clearance from burn wounds increases when Flt3L treatments begin early after burn injury. Burned mice were injected once daily with Flt3L or an equivalent volume of Lactated Ringers solution (LR) as a control treatment (n = 14 mice) for 4 days, as illustrated in Fig. 6. FL-0 treatment was initiated on the day of burn injury (n = 13 mice), FL-1 treatment was initiated 1 day after burn (n = 11 mice), and FL-3 treatment was initiated 3 days after burn (n = 13 mice). Three days after burn injury, wounds were inoculated with 8000 CFU P. aeruginosa and the inoculation region was harvested 72 h later for bacterial culture. Data show mean ± SEM in CFU per gram tissue wet weight. *, p < 0.05, significantly different from from Lactated Ringers solution (0 treatment in CFU/gram) group, by ANOVA Tukey’s multiple comparison test. Additionally, Pearson correlation R = –0.9886, p = 0.0114 by two-tailed, 95% confidence intervals.

 
Systemic bacterial clearance

To determine the effects of Flt3L treatments, independently of wound immunity, on systemic clearance of bacteria, mice were challenged with live P. aeruginosa by i.p. injection. Inoculation of mice was performed at 7 days postburn or sham injury to mimic the time frame at which systemic dissemination of bacteria from infected burn wounds typically occurs in our model. When burn wounds are inoculated at 3 days postburn with 8000 CFU P. aeruginosa, it takes ~3–4 days for apparent dissemination of P. aeruginosa into blood and tissues (data not shown). Therefore, clearance of an i.p. challenge was examined at 7 days postburn. Sham, control-treated mice had mean values of 5.4 x 104 CFU/ml blood and 1.1 x 107 CFU/gram in the spleen 16 h after inoculation, whereas burned, control-treated mice had 2.4 x 105 CFU/ml blood and 9 x 107 CFU/gram in the spleen (Fig. 8A). Although there were not significant differences between bacterial clearance in the burned and sham mice, the burned, control-treated mice tended to have a higher bacterial burden. However, burned mice treated with Flt3L for 4 days had mean values of only 629 CFU/ml blood and 8.9 x 103 CFU/gram in the spleen. Interestingly, Flt3L treatments did not alter bacterial clearance in the sham mice, who had mean values of 9.7 x 104 CFU/ml blood and 2.5 x 106 CFU/gram in the spleen.


Figure 8
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  		FIGURE 8. Clearance of a systemic bacterial challenge is enhanced in burned mice treated with Flt3L after burn injury. Seven days after burn or sham injury, mice were inoculated with 5 x 107 CFU P. aeruginosa by i.p. injection and tissues were harvested 16 h later for bacterial culture. Data show mean ± SEM in CFU/ml blood or gram tissue wet weight by log scale (n = 6–9 mice per group). A, Sham or burned mice were injected once daily with Flt3L or an equivalent volume of Lactated Ringers solution (LR) as a control treatment for 4 days, beginning on the day of sham or burn injury. B, Burned mice were injected once daily with Flt3L or an equivalent volume of Lactated Ringers solution as a control treatment for 4 days (X on timeline). Early Flt3L and control treatments were initiated on the day of burn injury and late Flt3L treatment was initiated 4 days after burn.

 
In a separate experiment, early and late Flt3L treatment protocols were compared (Fig. 8B). Control-treated mice had a mean value of 2.9 x 105 CFU/ml blood 16 h after inoculation, whereas early and late Flt3L-treated mice had mean values of only 21.7 and 398 CFU/ml blood, respectively. Similarly, control-treated mice had a mean of 1.3 x 108 CFU/gram in the spleen, whereas mice treated with FL, early and late after burn injury, had mean values of 2.9 x 103 and 7.9 x 105 CFU/gram in the spleen, respectively. Although the differences between groups were not statistically significant with this sample size (n = 6 mice/group), bacterial counts were from 1000 times to over 10,000 times lower in the mice that had been treated with Flt3L either late or early after burn injury, compared with control-treated burned mice.

Effect of Flt3L on Langerhans cells

Langerhans cells are DC that are present in large numbers in the epidermis and are an important immunological component of the interface between the host and the external environment. Because bacterial clearance from burn wounds correlated with the number of Flt3L treatments before wound inoculation, we examined the effects of Flt3L treatments on Langerhans cells in the skin bordering the burn wound. Langerhans cell numbers were not counted within the burn wound because a full-thickness injury was induced in this model. We therefore examined Langerhans cell numbers in the skin directly adjacent to the burn wound. Consistent with the literature, Langerhans cells were localized almost exclusively in the epidermis of normal skin from sham mice (data not shown). However, after burn injury Langerhans cells were also localized in the dermis of the skin bordering the wound. Flt3L treatments for 2 or 4 days did not have a significant effect on Langerhans (CD207+) cell numbers in wound-bordering skin 2 or 4 days after burn injury, respectively (Fig. 9). After 6 days of Flt3L treatments, there was a significant increase in the number of Langerhans cells in wound-bordering skin 7 days after burn injury (2.2 times more than in the control treatment (Lactated Ringers solution) group; p ≤ 0.05). Inoculation of burn wounds induced an even greater presence of Langerhans cells in the dermis of adjacent, uninjured skin. In control-treated mice Langerhans cell numbers were significantly (p ≤ 0.05), nearly 2-fold, greater 72 h after wound inoculation, compared with similar control-treated mice that did not receive wound inoculation. In mice that received 6 days of Flt3L treatment, 4 days of which were administered before wound inoculation, there was an even greater induction of Langerhans cells in wound-bordering dermis. Specifically, Flt3L-treated mice had 2.6 times more cells 72 h after inoculation than did the control-treated mice, and significantly more cells than all groups examined (p ≤ 0.05). Additionally, overall (CD207–) dermal cellularity appeared to be increased after infection in the Flt3L-treated mice, compared with control-treated mice (Fig. 9).


Figure 9
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  		FIGURE 9. Langerhan cell numbers in skin adjacent to burn wounds are steadily increased with daily Flt3L treatments, and further induced by wound infection. Burned mice were injected once daily with 10 µg of Flt3L or an equivalent volume of Lactated Ringers solution (LR) as a control treatment, beginning on the day of injury. Mice that were sacrificed 2 days after burn received 2 days of treatment; mice sacrificed 4 days after burn received 4 days of treatment, and those sacrificed 7 days postburn received 6 days of treatment. In the infection groups, mice were treated with Flt3L or Lactated Ringers solution for 6 days beginning on the day of injury. Wounds were inoculated (8000 CFU P. aeruginosa) 3 days postburn. Sections of burn wound and adjacent skin were stained with Abs specific for Langerin (CD207). Data show mean number ± SD of Langerin-positive cells per field examined under x40 objective. Langerin-positive cells in the dermis of adjacent, nonburned skin were quantitated. Three mice per group were included, with six separate counts performed from each mouse. #, p < 0.05; *, p < 0.05 significantly different from all other groups, using ANOVA and Tukey’s Multiple Comparison test. Representative photographs at magnification x400 show CD207 staining (bright pink) in dermis adjacent to burn wounds in mice from the infection groups.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Numerous reports have confirmed that severe burns can induce compromising alterations to the immune system. The immunological response to severe burns is complex, and appears to vary depending on the area, depth, and nature of the burns (flame or scald), and the presence of other insults such as smoke inhalation injury and nonthermal traumas (34). Additionally, the timing and adequacy of fluid resuscitation, wound excision and grafting, and nutritional status also appear to be important factors in determining the degree of immunological insult induced by burns (40). In addition to the potential for inadequate responses to infection, burn patients are at excessive risk for exposure to opportunistic organisms through the wound due to compromise in the barrier typically provided by the skin. Therefore, both systemic and local wound and skin immunity are important for controlling infections in burn patients.

DC are critical for regulating immune responses in the skin, as both sensors of immunological danger and activators of innate and acquired immune responses through Ag recognition, cytokine production, and presentation of Ags and costimulatory molecules to T cells. DC that reside in the epidermis, Langerhans cells, are the first line of defense against cutaneous infections and are typically identified by the expression of CD207, or Langerin. Dermal DC are typically distinguished from Langerhans cells by the absence of CD207 expression. Both epidermal and dermal DC are derived from flt3 receptor-positive progenitor cells and are increased in the skin following Flt3L treatments (41, 42, 43). Enhancement of Langerhans cell production by Flt3L is likely to be important in burn injury because Langerhans cells are replaced by flt3-positive bone marrow-derived progenitor cells during skin inflammation, whereas steady-state Langerhans cells are capable of self-renewal in the skin (43). Full-thickness injuries destroy the epidermal and dermal layers, so local control of wound infection is dependent upon cells migrating to the wound from other sources, possibly including the adjacent, intact skin. In this study, Langerhans cells were detected in the dermis of intact skin adjacent to the wound after a burn injury. Inoculation of burn wounds further increased the presence of Langerhans cells in the dermis bordering the wound, but the source and migration patterns of these cells could not be determined in this study. Daily administration of the DC growth factor Flt3L after burn injury caused a time-dependent increase in the number of Langerhans cells in the wound-bordering dermis. The significant, nearly 3-fold compared with control-treated mice, increase in Langerhans cell numbers in wound-bordering dermis following wound inoculation suggests that Flt3L increases the response to a wound infection. In addition to increased Langerhans cells at the borders of infected burn wounds of Flt3L-treated mice, overall cellularity was also increased. The cellular infiltrates were not identified, but could be neutrophils or macrophages, which play an important role in the inflammatory and debridement processes of wound healing (44), or other DC subtypes such as dermal DC. It is not clear from these studies whether the cells are migrating to the infection site, or from the site of infection in transit to the lymphatics. However, it has been reported that corneal Langerhans cells laterally migrate to a local thermal injury (45). Additionally, increased numbers of DC have been detected in the dermis at the border of human burn wounds (46). Under steady-state conditions Ags in the epidermis and dermis are trafficked to the draining lymph nodes only by TGF-β1-dependent cells, which are likely to be Langerhans cells (47).

The increased presence of various immune cells expressing high levels of CD69 in the wound draining lymph nodes suggests that increased mobilization of Langerhans cells, and likely other DC, near the infected burn wound results in enhancement of the overall immune response to infection. Surface expression of CD69 is increased on lymphocytes (48), and other immune cells (39, 49, 50), during the early stages of activation. This increased immune cell activation in Flt3L-treated mice is specifically in response to the infection because Flt3L treatments or burn injury alone did not increase the induction of CD69, CD25, IL-6, or cell death. Flt3L treatments by themselves do not directly activate the various effector cells examined because these cells do not express the Flt3 receptor due to down-regulation on progenitor cells during cellular commitment and differentiation (16). Surprisingly, CD69 expression was not induced by burn wound infection in mice that were not treated with Flt3L (Fig. 2), consistent with suppression of immune function after a severe burn injury. Although the model of burn wound infection excludes a direct comparison with an infection only, no-burn control, increased CD69 expression on immune cells is a typical response to infections (51). Unlike the control-treated mice, there was a significant increase in CD69+ cells in Flt3L-treated mice 72 h after wound inoculation. Significantly increased expression of CD69 in response to infection was observed on neutrophils, macrophages, B cells, and T cells of Flt3L-treated mice. Additionally, there was a significant increase in the proportion and number of T cells, but not B cells, expressing the activation marker CD25 (Fig. 3) in response to wound infection in Flt3L-treated mice. The lack of concomitant CD25 and CD69 induction in B cells in response to wound infection could possibly be due to the early time of harvest relative to systemic dissemination of bacteria. CD69 is very quickly up-regulated with immune activation in vivo, whereas CD25 induction is not as widespread and can be difficult to detect in vivo (52, 53, 54). The detection of CD25 on T cells suggests that T cell activation is either more predominant than, or precedes, B cell activation during a P. aeruginosa wound infection in Flt3L-treated mice. The presence of activated effector cells in the wound draining lymph nodes and spleens in response to a P. aeruginosa wound infection in the Flt3L-treated mice, together with increased numbers of CD11c+/class II MHChigh DC, suggests that Flt3L treatments after burn injury enhance the ability of the burned host to effectively respond to an infection. This is further supported by the apparent decreases in bacterial burden, and improved survival in the Flt3L-treated mice.

It is well established that DC rapidly undergo apoptosis during sepsis, and it has been proposed that DC depletion subsequently impacts lymphocyte function and survival (55, 56, 57). After a burn wound infection, there was a significant increase in leukocyte death in the spleen and wound draining lymph nodes (Fig. 4). There was no significant difference in the percentage of cells that was already dead at 72 h postwound inoculation. However, the percentage of cells undergoing early stages of apoptosis was significantly lower in mice that had been treated with Flt3L. This could be due to specific effects of Flt3L on cell survival or simply due to the relative increase in newly generated DC and hemopoietic precursors. The percentages of early apoptotic B cells and DC after wound infection were relatively high (compared with T cells) but consistent with other reports of apoptosis after immune stimulation (58, 59). Differences between control- and Flt3L-treated mice were not significantly different, although there was a trend toward decreased apoptosis in both cell types after Flt3L treatments. However, there was a significant increase in the percentage of apoptotic T cells after wound infection in the Flt3L-treated mice. Whereas the reason for this increase in T cell apoptosis is not known, it is possible that increased T cell activation in response to the wound infection, indicated by increased CD69 and CD25 expression in T cells of Flt3L-treated mice, may lead to increased activation-induced apoptosis in an attempt to prevent uncontrolled inflammation and tissue damage. The mechanisms and consequences of enhanced T cell apoptosis after infection in Flt3L-treated mice will be addressed in future studies. Overall, the beneficial effects of Flt3L on survival and bacterial clearance following burn wound infection suggest that enhanced T cell apoptosis does not negatively impact the postburn immune response to infection.

We found a strong correlation between the number of treatments given before wound inoculation and clearance of bacteria from wounds, suggesting that more time is required for Flt3L to affect local immunity in the skin (Fig. 7). Indeed, 6 days of treatment were necessary to see a significant increase in the number of Langerhans cells in the skin adjacent to burn wounds (Fig. 9). However, the systemic responses to infection appear to require less time or less treatment with Flt3L before infection. Because Flt3L treatments can be delayed, even until the day of wound inoculation, and remain effective in promoting survival (Fig. 6), Flt3L must have relatively rapid effects that enhance systemic responses to infection. In this model of burn wound infection mortalities in nontreated mice typically begin to occur 3–4 days after wound inoculation, which is when systemic infection becomes detectable in these mice. As a model of systemic dissemination of P. aeruginosa from the wound at 4 days postinoculation (7 days postburn), independent of wound clearance, we examined dissemination of P. aeruginosa following an i.p. challenge at 7 days postburn. Mice whose Flt3L treatments began either on the day of burn injury or 4 days after injury had fewer bacteria in their blood and spleens than did the control-treated mice (Fig. 8). Systemic clearance appeared to be slightly greater in the mice treated early, instead of later, with Flt3L, but bacterial burden was lowered by either course of Flt3L treatments. These data suggest that by the time bacteria would disseminate from the wound, Flt3L (started late or early after injury) has already mobilized and enhanced circulating DC such that systemic infection is rapidly cleared.

Surprisingly, Flt3L did not significantly alter bacterial clearance in sham, noninjured mice (Fig. 8). Bacterial burden in blood and spleens of control-treated mice following an i.p. challenge with P. aeruginosa was 4.4 to 8 times lower in sham, compared with burn-injured, mice. Flt3L did not change bacterial burden in the sham mice, whereas it enhanced clearance in the burned mice such that infection was almost eliminated by 16 h postinoculation. The reason for the lack of impact on bacterial clearance in sham mice is not known, but the data suggest that Flt3L may be most effective when immune status is compromised. However, further studies are required to address this issue. Nonetheless, Flt3L clearly has a significant impact on bacterial clearance mechanisms, both locally and systemically, following burn injury.

There have been numerous reports that burn injury predisposes the host to an excessive and potentially destructive inflammatory response to immunological stimuli, due to an increased capacity of monocytes or macrophages to produce inflammatory mediators, including IL-6 (60, 61, 62). We observed significantly high levels of IL-6 in the sera of control-treated mice 72 h after a burn wound inoculation. IL-6 levels after infection were significantly lower in the Flt3L-treated mice (Fig. 5), despite the concomitant increase in immune cell activation. This finding may be the result of decreased bacterial growth and spread, preventing a subsequent activation of systemic inflammation. Alternatively, because Flt3L promotes the large-scale production or enhancement of DC, perhaps burn-associated inflammatory monocytes are proportionately lower, so the relative impact of their response to infection is decreased. It has been reported that Flt3L-differentiated DC, compared with monocyte-derived DC, produce less IL-6 upon stimulation yet are equivalent in their capacity to stimulate T cell proliferation and to present Ag to CTLs (63). Because Flt3L treatments increase bacterial clearance in burned mice (Figs. 7 and 8), to determine whether Flt3L also alters the inflammatory response to bacterial challenge, systemic IL-6 was measured following a noninfectious i.p. challenge with HKPA. Overall levels of IL-6 were lower following HKPA challenge compared with wound infection. This decrease may be due to differences in the responses to live vs noninfectious bacterial challenge or due to differences in the responses to a bolus challenge vs a gradual and progressive infection, as occurs in a burn wound infection. IL-6 induction was depressed in burned mice, compared with noninjured mice, 7 days following burn injury (Fig. 5). Flt3L did not further decrease IL-6 levels in burned mice, but did decrease IL-6 in the sham mice. These data would suggest that Flt3L may influence inflammatory responses to bacterial stimuli both by controlling bacterial dissemination from the burn wound and by altering IL-6 induction. Future experiments will address the differential responses of burn and sham mice to bacterial challenge after Flt3L treatments.

In summary, Flt3L treatments after burn injury increase DC numbers both systemically and locally within the burn wound, and promote enhanced activation of numerous effector cells in response to a burn wound infection, without promotion of systemic inflammation. Flt3L treatments by themselves do not activate immune responses in the absence of infection, at least in terms of CD69 and CD25 expression and systemic IL-6 production. Treatment of burned mice with Flt3L after burn injury increases survival in an otherwise lethal model of burn wound infection, and treatment can be initiated early (day of burn injury) or late (day of wound inoculation) and remain effective at promoting survival. Whereas enhancement of local defense within the burn wound requires earlier treatments relative to induction of wound infection, enhancement of global immune responses to infection appears to be rapid, suggesting that Flt3L may also be effective at promoting resistance to other, nonwound originating infections after burn injury. The lack of a correlation between bacterial clearance from burn wounds and survival suggests that wound bacterial clearance is not the primary determinant of survival in this model. Nonetheless, the ability of early Flt3L treatments postburn to enhance local wound immunity and facilitate bacterial clearance from wounds is an important finding that may be beneficial in the clinical scenario in which burn wound infections are common sources of delayed wound healing and sepsis.

We previously reported that resistance to burn wound infection, in terms of survival, could be conferred to recipient mice by the adoptive transfer of isolated DC from Flt3L-treated, but not control-treated, donor mice (28), indicating that the protective effects of Flt3L are mediated by DC. Although expression of the Flt3 receptor is decreased during DC development, differentiated DC maintain a low level of Flt3 receptor expression, suggesting that Flt3L may have some direct effects on differentiated DC in addition to their hemopoietic precursors (16, 64). Together, these data support the hypothesis that Flt3L treatments indirectly enhance overall immune responses to a burn wound infection by increasing the number or functional capacity of DC to interact with and stimulate other effector cells of the immune response. Enhancement of DC production and immunostimulatory capacity with agents such as Flt3L may have clinical potential for prophylactic immunomodulation after burn injury to prevent infections in burn patients.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by research Grants R01 GM072810 from the National Institutes of Health and 8810 from the Shriners Hospitals for Children. Back

2 Address correspondence and reprint requests to Dr. Tracy Toliver-Kinsky, Department of Anesthesiology, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-0591. E-mail address: ttoliver{at}utmb.edu Back

3 Abbreviations used in this paper: Flt3, fms-like tyrosine kinase-3; Flt3L, Flt3 ligand; DC, dendritic cell; HKPA, heat-killed P. aeruginosa. Back

Received for publication November 26, 2007. Accepted for publication December 16, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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