INTRODUCTION
Severe trauma leads to a systemic inflammatory response caused by hormonal, metabolic, and immunological mediators, which are required for tissue repair. The trauma patient is exposed to several events or hits. The first hit is the trauma itself and the following hits are necessary surgeries performed with adverse events and/or complications during hospitalization, resulting in a weakened immune system with increased risk of infection and sepsis. In general, severe trauma is accompanied by massive blood loss leading to hemorrhagic shock. Sepsis that follows severe hemorrhagic shock often results in multiple organ dysfunction and death. Approximately 10% of multiply injured patients develop sepsis with a mortality rate of 18.2% during their hospital stay (1). When sepsis occurs, numerous redundant inflammatory cytokines are released into the blood stream, including tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) as well as the adipokine leptin (2). Leptin belongs to the family of long-chain helical cytokines, consisting of four interconnected antiparallel α-helices, and has structural similarity to IL-6 (3). It has a circadian rhythm with highest levels between 0 to 6 AM and lowest levels between 12 to 6 PM. Leptin is synthetized by differentiated mature adipocytes of white adipose tissue (4), it acts via the central nervous system, and modulates a variety of biological functions (5). It decreases appetite and increases energy expenditure, both resulting in weight loss (6), and raises hematopoiesis and lymphopoiesis in the bone marrow (7). Leptin has a pleiotropic role on the immune response. It exerts protective anti-inflammatory effects in models of acute inflammation and in processes activating innate immune responses (8). In contrast, leptin rather has a pro-inflammatory role during adaptive immune responses (8) that are typically divided into distinct CD4+ T-cell subtypes, T helper 1 (Th1, producing mainly pro-inflammatory cytokines) and Th2 (secreting modulatory and anti-inflammatory peptides). In vivo, leptin skews immune responses toward the Th1 phenotype and suppresses Th2 responses during immunosuppression induced by starvation (9).
So far, clinical studies have yielded inconsistent results, suggesting a rather complex role for leptin in immune-mediated inflammatory conditions in humans (8). In 1998, Bornstein et al. (10) and in 1999, Arnalich et al. (11) found that non-trauma patients suffering from severe sepsis had higher leptin concentrations than the chosen control groups. Furthermore, they observed higher leptin concentrations in survivors of severe sepsis than in non-survivors. In murine pneumonia models, leptin deficiency resulted in a weakened immune defense, benefits for the host defense in these models were observed after leptin substitution (12). Noteworthy, in patients with sepsis both a negative (10, 11) and a positive (13) correlation between leptin and IL-6 have been reported by researchers.
In view of the data available, the objective of our study was to evaluate a possible, dose-dependent therapeutic impact of exogenous leptin on mortality, lymphocyte populations, cytokine serum levels, extent of organ damages, type IV delayed hypersensitivity reaction, body weight, body temperature, and activity level in a trauma/sepsis model consisting of a femur fracture, hemorrhagic shock, and subsequent sepsis. In addition, we wanted to investigate if changes caused by exogenous leptin were related to the presence of IL-6. Therefore, we repeated the test series with IL-6 knockout (IL-6−/−) mice to elucidate the role of IL-6 in the therapeutic actions and pathogenetic pathway of leptin administration.
MATERIALS AND METHODS
The study was approved by the local legislative committee (Nr. 7602) and conducted according to the “Guide for the Care and Use of Laboratory Animals of the National Institute of Health.” Below, the study design is briefly presented, whereas detailed information is provided as supplemental material.
Animals
The experiments were performed in 69 wild-type mice of the inbred strain C57BL/6 and 63 IL-6−/− mice of the inbred strain B6.129S2-IL6tm1/Kopf/J. Their mean body weight was 20.0 ± 2.9 g and their mean age was 8 weeks. IL-6 deficiency was proven by an ELISA test in the serum of each IL-6−/− mouse prior to the test series. Wild-type mice were randomly divided into nine subgroups, as presented in Table 1. In the same way, nine groups of IL-6−/− mice with at least six animals were formed. All procedures were conducted after deeply anesthetizing the animals (see supplemental data, https://links.lww.com/SHK/A552). Every day, each mouse was checked for signs of pain and if needed, an escalating analgesic regime would have been used. However, this was not necessary in our study.
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Table 1: Randomization of wild-type mice into nine subgroups
A time chart is presented in Figure 1. On day 2, the back skins of the mice were shaved and sensitized with 50 μL 1% 2.4-dinitrofluorobenzene (DNFB; Sigma-Aldrich Chemie GmbH, Steinheim, Germany) in acetone/olive oil (4:1). On day 0, a standardized closed fracture (AO-classification A2) of the right femur was induced in all mice assigned to the trauma groups and trauma/sepsis groups, using a blunt guillotine device modified according to Hiltunen et al. (14) with a weight of 520 g. After closed reduction, the leg was splinted. The orbital plexus was punctured to withdraw 60% of the total blood volume, hence inducing a hemorrhagic shock. After 60 min of hypovolemia, reperfusion via the tail vein was performed using sterile ringer lactate, four times as much as the extracted amount of blood. On day 2 (48 h after induction of fracture and hemorrhage), all mice of the trauma/sepsis groups were anesthetized for the second time to induce polymicrobial sepsis using a cecal ligation and puncture (CLP) method. CLP represents a peritonitis model with clinical features of polymicrobial infection consistent with those of peritonitis in humans (15). Directly after sepsis induction or laparotomy, recombinant murine leptin (PEPRO Tech Inc, Rocky Hill, Conn) dissolved in 0.9% saline was applied in a dose of 2.5 μg/g body weight or 5 μg/g body weight. Animals of the vehicle groups received only 0.9% saline solution. On day 5, the dorsal surface of the right ear of each mouse was challenged with 50 μL 0.5% DNFB. Finally, on day 6 (144 h after induction of fracture and hemorrhage), a relaparotomy was performed under general anesthesia and the mice were exsanguinated by direct cardiac puncture. Heart, lung, liver, spleen, and kidneys were excised.
Fig. 1: Time flow of procedures applied to the mice of the trauma/sepsis groups.CLP indicates cecal ligation and puncture.
Evaluation of body parameters and vital signs
Body temperature, body weight, activity level, and ear thickness were documented each morning at the same time over the 8-day study period. The activity level was rated on a scale of 1 to 6, based on defined behavioral characteristics as described below. 1 (moribund): no activity, reduced vital functions, death is expected; 2 (lethargic): no activity, remaining in the same position, no food intake; 3 (quiet): sporadic activity, indifferent toward the environment, sleepy; 4 (limitedly active): attentive, many breaks in activities; 5 (active): curious, fast, sporadic breaks in activity; 6 (very active): strong, curious, fast movements. The immune-modulatory effect of leptin was quantified by measuring hypersensitivity type IV reaction according to Coombs and Gell (16) and assessed as tissue swelling of the right ear. The difference of the ear thickness, measured with a spring-loaded micrometer (Oditest, Dresden, Germany) immediately prior to challenge and 24 h after challenge, was used for analysis.
Cytokine determination
To investigate the impact of leptin treatment on trauma- and sepsis-related cytokines in the blood, tumor necrosis factor-α (TNFα), monocyte chemoattractant protein-1 (MCP1), IL-6, IL-10, IL-12p70, and interferon γ were quantified in the plasma with the Cytometric Bead Array (CBA) Mouse Inflammation Kit (BD Biosciences Pharmingen, San Diego, Calif). All of the cytokines mentioned above were measured in one process from one single sample of 50 μL. Data received were analyzed with the BD CBA Analysis Software; cytokine concentrations were determined by means of a standard curve.
Flow cytometry of lymphocytes from the circulation and from the spleen
Cellular material was examined for the cell surface antigens CD4+ of T-helper cells, CD8+ of cytotoxic T-cells, as well as Ly49+ of natural killer cells. To identify these antigens, the following fluorochrome-conjugated monoclonal antibodies were added: antimouse CD4 antibodies + fluorescein isothiocyanate (FITC), antimouse CD8 antibodies + phycoerythrin (PE), and antimouse LY49 antibodies + FITC. After washing, the analysis was performed using a FACSCalibur Flow Cytometer (fluorescence-activated cell scanner, Becton Dickinson, Heidelberg, Germany). Of each sample, 5,000 cells were counted. Data analysis was performed with WinMDI 2.8. To identify apoptotic cells, an assay kit with annexin V conjugated to FITC and propidium iodide (PI) was used. Annexin V-positive/PI-negative staining was regarded as apoptosis and PI-positive staining as necrosis.
Histopathology
The histologic samples were tested for pathologic tissue changes with a light microscope (Carl Zeiss, Jena, Germany). Each assessment was based on 10 high-power microscopic fields of view. Referring to lung and liver, interstitial thickening and infiltration of neutrophil granulocytes were rated. Additionally, hydropic cell degeneration was investigated in the liver. Necrosis, filling level of white pulp with lymphocytes, and separation of red and white pulp of the spleen were evaluated. The kidney was scanned for infiltration of neutrophil granulocytes and filling level of the glomeruli with mesangial cells. The relevant scoring systems are presented in Table 2.
Table 2: Scoring system
Statistical analysis
Data were analyzed using Microsoft Excel 2013 IBM, SPSS Statistics Version 21, 64-bit and Sigmastat 3.5 (SPSS Inc, Armonk, NY). According to the distribution, data were expressed as mean ± standard error of the mean or as median and interquartile range (in round brackets). P values lower than 0.05 were considered to be statistically significant. The Kolmogorow–Smirnow test was performed to assess normal distribution of the samples. To compare three groups, we performed one-way analysis of variance for normally distributed samples and Kruskal–Wallis tests for non-normally distributed samples. In case of significant differences, Tukey method as a post hoc test for pairwise comparison was used for normally distributed samples, whereas Dunn test was performed for non-normally distributed samples. Categorical data were analyzed by means of the chi-square test. Differences in survival were determined by the log-rank test performed on Kaplan–Meier curves.
RESULTS
Mortality in wild-type mice and IL-6−/− mice
All wild-type mice and IL-6−/− mice in the trauma groups and sham groups survived, whereas four mice (36.4%) in the wild-type-trauma/sepsis-vehicle group, three mice (25%) in the wild-type trauma/sepsis leptin1 group, and not a single mouse in the wild-type trauma/sepsis leptin2 group died. Figure 2A presents the relevant Kaplan–Meier curves of the wild-type trauma/sepsis group. Interestingly, a leptin dose of 5 μg/g body weight significantly decreased the mortality rate of the wild-type mice (P = 0.034). In contrast, leptin administration increased the mortality rate of the IL-6−/− mice with increasing leptin dose (IL-6−/− trauma/sepsis vehicle group: 53.8%, IL-6−/− trauma/sepsis leptin1 group: 83.4%, IL-6−/− trauma/sepsis leptin2 group: 100%), as presented in Figure 2B. However, a significant difference in mortality was only calculated between the IL-6−/− trauma/sepsis vehicle group and the IL-6−/− trauma/sepsis leptin2 group (P = 0.032).
Fig. 2: Survival rate.Kaplan–Meier curves (A) of the wild-type trauma/sepsis group and (B) of the IL-6−/− trauma/sepsis group. First hit: induction of a standardized closed fracture of the right femur on day 0. Second hit: induction of sepsis using CLP on day 2. Leptin1: 2.5 μg/g body weight. Leptin2: 5 μg/g body weight. Vehicle: 0.9% saline solution. CLP indicates cecal ligation and puncture.
BODY PARAMETERS AND VITAL SIGNS IN WILD-TYPE MICE
Trauma/sepsis group
Body temperature decreased in each trauma/sepsis group after CLP on day 2. Figure 3A graphically displays the change in body temperature, based on the initial value at test start. No significant differences between any pair of groups could be revealed. The decline of activity level was also triggered by CLP. Figure 3B shows an almost identical course for the three groups. Body weight was recorded as difference between the actual body weight and the body weight measured after CLP (Fig. 3C). Weight loss in the trauma/sepsis leptin2 group was higher on days 4, 5, and 6 compared with the trauma/sepsis vehicle-group (P ≤ 0.018). Leptin administration inhibited ear swelling in septic mice, as Figure 3D shows. Type IV-delayed hypersensitivity was less pronounced in the trauma/sepsis leptin1 group and trauma/sepsis leptin2 group compared with the trauma/sepsis vehicle group (P = 0.039; P = 0.018).
Fig. 3: Body parameters and vital signs.Change in (A) body temperature, (B) activity level, (C) body weight of wild-type trauma/sepsis mice (* denotes a significant difference between the leptin2 and the vehicle group). D, Increase in ear thickness of all wild-type mice (# denotes a significant difference between the trauma/sepsis vehicle group and the corresponding leptin1 and leptin2 group). First hit: induction of a standardized closed fracture of the right femur on day 0. Second hit: induction of sepsis using CLP on day 2. Leptin1: 2.5 μg/g body weight. Leptin2: 5 μg/g body weight. Vehicle: 0.9% saline solution. CLP indicates cecal ligation and puncture.
Sham group and trauma group
During the observation period, body temperatures and activity levels were almost similar in all sham groups and trauma groups. Immediately after laparotomy, wild-type mice in all six groups lost weight, but gained it again. In contrast to the trauma/sepsis group, leptin in the dose of 5 μg/g bodyweight increased the ear swelling in the sham model (sham leptin2 group: 0.15 ± 0.03 μm, sham vehicle group: 0.035 ± 0.01 μm; P = 0.018), whereas no clear tendencies were apparent in the trauma group.
Comparison of the three groups
On days 3 to 6, body temperature was lower in the trauma/sepsis vehicle group compared with the sham vehicle group (P ≤ 0.018) and compared with the trauma vehicle group (P ≤ 0.016). Starting on day 3, the activity level was lower in the trauma/sepsis mice than in the sham mice and trauma mice (P < 0.0001). In the trauma/sepsis vehicle group, weight loss was higher on days 5 and 6 compared with the sham vehicle group (P ≤ 0.020) and on day 6 also compared with the trauma vehicle group (P = 0.006). Ear swelling was more pronounced in wild-type mice of the trauma/sepsis vehicle-group (0.26 ± 0.04 μm) compared with the sham vehicle group (0.065 ± 0.02 μm; P = 0.02).
CYTOKINES IN WILD-TYPE MICE
Trauma/sepsis group
The cytokine levels detected in the wild-type trauma/sepsis group are presented in Table 3. Higher TNFα levels were found in the vehicle group, whereas leptin prevented an increase in TNFα, with no significant differences as a result of the strongly varying individual values. Leptin administration did not cause significant changes in MCP1 and IL-6 plasma levels. However, clear tendencies toward lower IL-6 levels were observed with increasing leptin dose. IL-12p70 was below detection level in the vehicle group. IL-10 was identified only sporadically, and INFγ could not be detected in general.
Table 3: Cytokine levels in wild-type mice of the trauma/sepsis group* presented as mean and standard deviation and† as median and interquartile range
Sham group and trauma group
TNFα was detected only in low concentrations in the sham group and the trauma group. Leptin administration did not cause significant changes in MCP1, IL-6, and IL-12p70 levels. IL-10 and INFγ could not be identified at all.
Comparison of the three groups
Higher MCP1 levels were found in the trauma/sepsis vehicle group compared with the sham vehicle group (22 (16-31) pg/mL; P = 0.036) and higher IL-6 values were detected in the trauma/sepsis vehicle group compared with the trauma vehicle group (93 ± 14 pg/mL; P = 0.023). Although leptin administration resulted in a decrease of MCP1 levels in the trauma group, it provoked an increase in the sham group and trauma/sepsis group, as Figure 4A displays. Leptin administration stimulated the production of IL-6 in the sham group, but it resulted in a decrease of IL-6 levels in the trauma groups and trauma/sepsis groups, which did not reach statistical significance (Fig. 4B).
Fig. 4: Cytokine levels.A, MCP1 levels. B, IL-6 levels in wild-type mice. Sham group: laparotomy; trauma group: first hit + laparotomy; trauma/sepsis group: first hit + laparotomy. First hit: induction of a standardized closed fracture of the right femur on day 0. Second hit: induction of sepsis using CLP on day 2. Leptin1: 2.5 μg/g body weight. Leptin2: 5 μg/g body weight. Vehicle: 0.9% saline solution. CLP indicates cecal ligation and puncture; IL, interleukin; MCP, monocyte chemoattractant protein.
CIRCULATING LYMPHOCYTES OF WILD-TYPE MICE
Trauma/sepsis group
The relative numbers of selected lymphocytes in the blood of wild-type mice suffering trauma and sepsis are presented in Table 4.
Table 4: Relative lymphocyte number in the blood of wild-type mice of the trauma/sepsis group
Sham group and trauma group
Compared with an extremely small number in the sham leptin1 group, necrotic cells were more frequently found in the sham vehicle group (1.6% ± 0.4%; P = 0.01) and in the sham leptin2 group (0.2 ± 0.2%; P = 0.015). With regard to the trauma group, no significant differences were detected.
Comparison of the three groups
CD4+ lymphocytes were less frequently found in the sham vehicle group (12.0% ± 0.6%) compared with the trauma vehicle group (21.0% ± 2.1%; P = 0.037). The number of CD8+ lymphocytes was higher in the trauma/sepsis vehicle group (P = 0.009) compared with the sham vehicle group (13.4% ± 1.4%). Finally, the number of necrotic cells was higher in the sham vehicle group compared with the trauma vehicle group (0.01% ± 0.00%; P = 0.016) and the trauma/sepsis vehicle group (P = 0.009).
SPLENIC LYMPHOCYTES OF WILD-TYPE MICE
Trauma/sepsis group
Relevant relative lymphocyte numbers in the spleen of septic wild-type mice are displayed in Table 5.
Table 5: Relative lymphocyte number in the spleen of wild-type mice of the trauma/sepsis group
CD4+ levels were higher in the trauma/sepsis leptin1 group than in the trauma/sepsis vehicle group (P = 0.006). The number of CD8+ lymphocytes was lower in the trauma/sepsis vehicle group compared with the trauma/sepsis leptin1 group (P = 0.005) and compared with the trauma/sepsis leptin2 group (P = 0.009).
Sham group and trauma group
In the sham group, differences in CD4+ lymphocyte numbers were revealed between the sham vehicle group (5.5% ± 0.7%) and the sham leptin1 group (13.8% ± 1.0%; P = 0.003). CD8+ percentages of the sham vehicle group (27.9% ± 2.4%) were higher compared with the sham leptin1 group (13.2% ± 0.7%; P = 0.024) and compared with the sham leptin2 group (12.7% ± 1.0%; P = 0.032). Also, Ly49+ levels were higher in the sham vehicle group (11.4% ± 3.2%) than in the sham leptin1 group (4.8% ± 0.4%; P = 0.002). Compared with the sham vehicle group (0.5% ± 0.1%), the number of necrotic cells was lower in the sham leptin1 group (0.02% ± 0.01%; P = 0.030) and in the sham leptin2 group (0.02% ± 0.01%; P = 0.018). With regard to the trauma group, no significant differences were revealed.
Comparison of the three groups
Compared with the trauma/sepsis vehicle group, lower CD4+ levels (P = 0.008) and higher CD8+ levels (P = 0.010) were revealed in the sham vehicle group. The number of Ly49+ lymphocytes was higher in the sham vehicle group than in the trauma group (4.6 ± 0.4; P = 0.011). Whereas necrosis was not detected in the trauma vehicle mice, a higher number of necrotic cells were found in the sham vehicle mice (P = 0.002) and in the trauma/sepsis vehicle mice (P = 0.014).
ORGAN DAMAGE IN WILD-TYPE MICE
Lung
Within the trauma/sepsis group, no significant differences in interstitial thickening and infiltration of granulocytes were observed. Abundant manifestation of both factors was rare, as displayed in Table 6. In the sham group, almost no interstitial thickening and infiltration of granulocytes was detected. However, interstitial thickening and infiltration of granulocytes were rated “existent” in 15.8% and 10.5% of the trauma mice. Comparing the three vehicle groups, a tendency toward an increase of both factors was revealed in the histological evaluation. The sham vehicle group had the lowest intensities, the trauma vehicle group had higher intensities, and finally the trauma/sepsis vehicle group had the highest values.
Table 6: Number of surviving wild-type mice of the trauma/sepsis group with an organ score of 0, 1, and 2
Liver
Within the trauma/sepsis group, no significant differences in interstitial thickening, infiltration of granulocytes, and hydropic degeneration could be detected (Table 6). Surprisingly, granulocytes were found neither in any of the three trauma/sepsis groups nor in any of the sham groups and trauma groups. No differences in the degree of interstitial thickening were observed within the sham group and within the trauma group. In both groups, hydropic degeneration was detected only in a small amount. Comparing the three vehicle groups, manifestation of interstitial thickening was almost similar, whereas hydropic degeneration was more pronounced in the trauma/sepsis vehicle group compared with the sham vehicle group (P = 0.031).
Spleen
The evaluation of the histological specimen of the spleen did not reveal any significant or tendential differences. In each of the nine groups, necrosis was not detected at all. Red and white pulpa were well differentiated and white pulpa were well filled.
Kidney
In the histological specimen, no significant or tendential differences were revealed either. An infiltration of granulocytes could not be detected and the glomeruli of all groups were well filled with mesengial cells.
COMPARISON OF WILD-TYPE MICE AND IL-6−/− MICE
Regrettably, we were unable to perform any further comparative tests planned for septic IL-6−/− mice due to the fact that solely one mouse in the IL-6−/− trauma/sepsis leptin1 group and not a single mouse in the IL-6−/− trauma/sepsis leptin2 group had survived for more than 96 h.
With regard to body temperature, activity level, and body weight, no significant differences between corresponding wild-type groups and IL-6−/− groups of the sham group and trauma group were observed. Ear swelling was less pronounced in the IL-6−/− trauma groups (wild-type trauma vehicle group: 0.23 ± 0.04 μm, IL-6−/− trauma vehicle group: 0.09 ± 0.03 μm, P = 0.014; wild-type trauma leptin1 group: 0.22 ± 0.06 μm, IL-6−/− trauma leptin1 group: 0.027 ± 0.01 μm, P = 0.002; wild-type trauma leptin2 group: 0.13 ± 0.06 μm, IL-6−/− trauma leptin2 group: 0.025 ± 0.01 μm, P = 0.015). Comparing the cytokine levels, we revealed a significant difference only for MCP1 (wild-type sham leptin2 group: 225 ± 6 pg/mL, IL-6−/− sham leptin2 group: 36 ± 9 pg/mL). Significant differences in the relative number of serum and splenic lymphocytes are presented in Table 7. Interestingly, the relative numbers of CD4+, CD8+, and finally, histologic examination did not provide significant differences in interstitial thickening and infiltration of granulocytes observed in lung and liver as well as in hydropic degeneration of the liver.
Table 7: Significant differences in lymphocyte numbers between wild-type mice and IL-6−/− mice
DISCUSSION
Globally, sepsis is reported to have an incidence of 56 to 91 cases per 100,000 people on average, with a reported mortality rate up to 30% for sepsis, 50% for severe sepsis, and 80% for septic shock (17). Despite many years of extensive research and numerous clinical studies, sepsis remains a major challenge both for clinicians and researchers. Its pathophysiology is still incompletely understood, because it can be characterized as a complex and dynamic disease process. The early stages of sepsis are associated with a potentially fatal hyperinflammatory state mediated by pro-inflammatory cytokines (18). As sepsis progresses, the immunologic response shifts to a hypo-inflammatory response, which results in an immunosuppressive state (19).
In general, a comparison of the three vehicle groups of the same inbred strain revealed the effects of uncomplicated surgery, of trauma and uncomplicated surgery, and of trauma followed by surgery and sepsis induction in mice left untreated with leptin. The fact that all mice in the sham vehicle group and trauma vehicle group of both wild-type mice and IL-6−/− mice survived, whereas fatalities exclusively occurred in both trauma/sepsis vehicle groups, provided evidence that our animal model was appropriate to simulate the two-hit model with trauma and subsequent sepsis. When comparing the three groups within a model, the impact of leptin (applied to mice at a dose of 2.5 μg/g body weight and of 5 μg/g body weight) was evaluated on a selected parameter in this certain model, in most cases pointing out significant differences or tendencies toward differences. The major findings of our study were that leptin administration in sufficient dosages significantly decreased the mortality of septic wild-type mice, that it did not negatively affect healthy mice and mice suffering from femur fractures, and that IL-6 had to be considered an important prerequisite for the protective effect of leptin on sepsis mortality.
Prior animal studies demonstrating a role of leptin in sepsis yielded conflicting results. Tschöp et al. (5) reported that central nervous system leptin optimized the immune response and increased survival in wild-type mice as well as in leptin-deficient mice after CLP, whereas Shapiro et al. (20) found that the continuous administration of leptin in wild-type mice over a time period of 7 days resulted in significantly lower survival rates after CLP. The main pathophysiological feature of sepsis is the uncontrollable inflammatory response as a consequence of the priming of immune effector cells by the first insult (21) resulting in an increase in both pro- and anti-inflammatory cytokines in the blood stream (22). By activating monocytes, leptin is known to dose-dependently stimulate the production of pro-inflammatory cytokines such as TNFα in vitro(23). Our results indicated the impact of leptin on this cytokine. As inflammation continued due to sepsis, TNFα values were considerably (though not significantly) higher in the trauma/sepsis vehicle group, whereas they were suppressed in the trauma/sepsis leptin1 group and in the trauma/sepsis leptin2 group. The decline in TNFα levels might have been the reason for the protective effect of leptin, resulting in a lower mortality rate of wild-type mice. With regard to TNFα, our results are in line with those of Koca et al. (24). They investigated leptin administration to rodents, which were exposed to live bacteria. Leptin raised TNFα levels in the control group, whereas they were reduced in infected animals.
Increased levels of MCP1 have been reported in septic patients (25) and in septic mice (26). Our values confirm these findings. Significantly higher MCP1 levels were detected in the trauma/sepsis vehicle group than in the sham vehicle group. The same pertained to IL-6. IL-6 is classified as both a pro-inflammatory and anti-inflammatory cytokine; it is not only involved in inflammation and infection responses, but also in the regulation of metabolic, regenerative, and neural processes (27). IL-6 is likely to be an important mediator of the inflammatory response in sepsis, because IL-6 levels were significantly higher in septic patients, who subsequently died than in those who survived (10, 11). In our test series of wild-type mice, leptin administration increased IL-6 levels in the sham mice, but it decreased IL-6 levels in the trauma mice and the trauma/sepsis mice, though without significant differences. Furthermore, the mortality rates show that leptin is IL-6-dependent. In wild-type mice suffering from trauma and sepsis, a leptin dose of 5 μg/g body weight significantly decreased the mortality rate, not a single mouse died. However, the administration of leptin in a dose of 5 mg/g body weight significantly increased the mortality rate, resulting in the death of all IL-6−/− mice that experienced trauma and sepsis.
Until today, only a few studies have focused on the interaction between leptin and lymphocytes. With regard to CD4+ T-cells, immunoregulatory properties of leptin referring to survival and proliferation have been reported (28). Furthermore, leptin deficiency was associated with a reduced number of circulating CD4+ T-cells that was reversed by recombinant leptin administration (29). Whereas we observed CD4+ levels in the blood of wild-type mice between the trauma/sepsis-leptin1-group and the trauma/sepsis leptin2 group which were only higher by tendency as compared with the trauma/sepsis vehicle group, we observed an increase of splenic CD4+ T-cells after leptin administration in a dose of 2.5 μg/g body weight in the sham model and in the trauma/sepsis model. The role of leptin in the maturation of Ly49+ NK cells has already been confirmed by their decreased number in leptin receptor-deficient mice (30). In accordance with this finding, our study indicated a significant dose-dependent impact of leptin on the number of splenic Ly49+ NK cells in wild-type mice of the sham model.
In general, leptin administration resulted in the activation of the immune system in the sham groups, whereas leptin administration after trauma and especially after trauma and sepsis induction led to the suppression of the overactivated immune system, as also indicated by the type IV hypersensitivity reaction. Whereas leptin in a dose of 5 μg/g body weight raised ear swelling in the sham model of wild-type mice, leptin administration in both doses inhibited ear swelling in septic wild-type mice.
Ninety percent of patients suffering from a severe sepsis are expected to have an increased body temperature (31), while a decline in body temperature after CLP was reported in rodents (32). Our results confirm the latter observation. Body temperature started to decrease after CLP in all groups of our trauma/sepsis model. However, exogenous leptin did not result in a rise in body temperature as reported by Pelleymounter et al. (33), probably caused by the fact that leptin was administered only once to septic mice and not in a continuous way. Leptin plays a crucial role in the homeostasis of body weight by regulating food intake and energy expenditure (34). Administration of leptin by injection or subcutaneous infusion has been reported to result in a dose-dependent decrease in body weight at incremental increases of plasma leptin levels within the physiological range (33, 34). In accordance with these findings, significantly higher values of weight loss in leptin-administered mice were revealed in our test series.
Summing up, our study revealed a dose-dependent anti-inflammatory effect of exogenous leptin in the trauma/sepsis group, whereas leptin showed to some extent a pro-inflammatory effect in the sham group. These apparently contradictory results may suppose that leptin influences the immunologic pathways corresponding to the underlying medical condition in an immunomodulatory way. To our knowledge, this is the first study that shows the protective impact of leptin on mortality in a two-hit trauma and sepsis model, depending on the presence of IL-6. The ability of leptin to beneficially modulate inflammation and the host response of mice suffering from sepsis might support its use as a therapeutic agent for the prevention or treatment of sepsis after initial trauma. Undoubtedly, further studies are necessary to confirm this hypothesis and to evaluate the interaction between leptin and IL-6.
Acknowledgment
The authors thank Fritz Seidl, MA Interpreting and Translating, for the linguistic correction of the paper.
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