Obesity Prolongs the Inflammatory Response in Mice After Severe Trauma and Attenuates the Splenic Response to the Inflammatory Reflex.

Thoracic traumas with extra-thoracic injuries result in an immediate, complex host response. The immune response requires tight regulation and can be influenced by additional risk factors such as obesity, which is considered a state of chronic inflammation. Utilizing high-dimensional mass and regular flow cytometry, we define key signatures of obesity-related alterations of the immune system during the response to the trauma. In this context, we report a modification in important components of the splenic response to the inflammatory reflex in obese mice. Furthermore, during the response to trauma, obese mice exhibit a prolonged increase of neutrophils and an early accumulation of inflammation associated CCR2+CD62L+Ly6Chi monocytes in the blood, contributing to a persistent inflammatory phase. Moreover, these mice exhibit differences in migration patterns of monocytes to the traumatized lung, resulting in decreased numbers of regenerative macrophages and an impaired M1/M2 switch in traumatized lungs. The findings presented in this study reveal an attenuation of the inflammatory reflex in obese mice, as well as a disturbance of the monocytic compartment contributing to a prolonged inflammation phase resulting in fewer phenotypically regenerative macrophages in the lung of obese mice.

May the neutrophil-to-lymphocyte ratio at admission predict the occurrence and the severity of ARDS after blunt chest trauma patients? A retrospective study.

INTRODUCTION: In blunt chest trauma patients, the activation of inflammatory response is thought to be one of the pathophysiological pathways leading to delayed acute respiratory distress syndrome(ARDS). The main objective of the study was to assess the performance of the neutrophil-lymphocyte ratio(NLR) for prediction of delayed ARDS. The secondary objective was to compare NLR in patients with traumarelated focal and non-focal ARDS. METHODS: Over a 2-year period, every adult patient triaged to our level 1 trauma center with multiple rib fractures and PaO 2 /FiO 2 ratio > 200 at admission were retrospectively included. The NLR was recorded at admission in the Emergency Department(ED). The main study outcome was the occurrence of moderate to severe ARDS within 5 days after admission according to Berlin criteria. Two phenotypes (focal and non-focal ARDS) were determined based on the closest chest CT regarding the ARDS onset. RESULTS: 216 patients were included and 42(19%) underwent moderate to severe ARDS within 5 days after ED admission (focal, N = 26 [12%] and non-focal, N = 16 [7%]). The NLR at ED admission was not statistically different between patients who developed or not a delayed ARDS (14 ± 13 vs. 11 ± 8,p = 0.095), although patients with non-focal ARDS presented higher NLR ratio than focal ARDS (21 ± 18 p < 0.0001). The AUC for NLR at ED in predicting delayed ARDS was 0.53. CONCLUSION: In blunt chest trauma patients, the NLR at ED admission was unable to predict delayed ARDS over the five first days post-injury. Although not clinically relevant, the NLR was higher in patients with non focal ARDS.

Intact Glucocorticoid Receptor Dimerization Is Deleterious in Trauma-Induced Impaired Fracture Healing.

Following severe trauma, fracture healing is impaired because of overwhelming systemic and local inflammation. Glucocorticoids (GCs), acting via the glucocorticoid receptor (GR), influence fracture healing by modulating the trauma-induced immune response. GR dimerization-dependent gene regulation is essential for the anti-inflammatory effects of GCs. Therefore, we investigated in a murine trauma model of combined femur fracture and thoracic trauma, whether effective GR dimerization influences the pathomechanisms of trauma-induced compromised fracture healing. To this end, we used mice with decreased GR dimerization ability (GRdim). The healing process was analyzed by cytokine/chemokine multiplex analysis, flow cytometry, gene-expression analysis, histomorphometry, micro-computed tomography, and biomechanical testing. GRdim mice did not display a systemic or local hyper-inflammation upon combined fracture and thorax trauma. Strikingly, we discovered that GRdim mice were protected from fracture healing impairment induced by the additional thorax trauma. Collectively and in contrast to previous studies describing the beneficial effects of intact GR dimerization in inflammatory models, we report here an adverse role of intact GR dimerization in trauma-induced compromised fracture healing.

Contributing factors in the development of acute lung injury in a murine double hit model.

OBJECTIVES: Blunt chest (thoracic) trauma (TxT) is known to contribute to the development of secondary pulmonary complications. Of these, acute lung injury (ALI) is common especially in multiply injured patients and might not only be due to the direct trauma itself, but seems to be caused by ongoing and multifactorial inflammatory changes. Nevertheless, the exact mechanisms and contributing factors of the development of ALI following blunt chest trauma are still elusive. METHODS: 60 CL57BL/6N mice sustained either blunt chest trauma combined with laparotomy without further interventions or a double hit (DH) including TxT and cecal ligation puncture (CLP) after 24 h to induce ALI. Animals were killed either 6 or 24 h after the second procedure. Pulmonary expression of inflammatory mediators cxcl1, cxcl5, IL-1ß and IL-6, neutrophil infiltration and lung tissue damage using the Lung Injury Score (LIS) were determined. RESULTS: Next to a moderate increase in other inflammatory mediators, a significant increase in CXCL1, neutrophil infiltration and lung injury was observed early after TxT, which returned to baseline levels after 24 h. DH induced significantly increased gene expression of cxcl1, cxcl5, IL-1ß and IL-6 after 6 h, which was followed by the postponed significant increase in the protein expression after 24 h compared to controls. Neutrophil infiltration was significantly enhanced 24 h after DH compared to all other groups, and exerted a slight decline after 24 h. LIS has shown a significant increase after both 6 and 24 h compared to both control groups as well the late TxT group. CONCLUSION: Early observed lung injury with moderate inflammatory changes after blunt chest trauma recovered quickly, and therefore, may be caused by mechanical lung injury. In contrast, lung injury in the ALI group did not undergo recovery and is closely associated with significant changes of inflammatory mediators. This model may be used for further examinations of contributing factors and therapeutic strategies to prevent ALI.

Immunostimulatory functions of adoptively transferred MDSCs in experimental blunt chest trauma.

Myeloid-derived suppressor cells (MDSCs) expand during inflammation and exhibit immunomodulatory functions on innate and adaptive immunity. However, their impact on trauma-induced immune responses, characterized by an early pro-inflammatory phase and dysregulated adaptive immunity involving lymphocyte apoptosis, exhaustion and unresponsiveness is less clear. Therefore, we adoptively transferred in vitro-generated MDSCs shortly before experimental blunt chest trauma (TxT). MDSCs preferentially homed into spleen and liver, but were undetectable in the injured lung, although pro-inflammatory mediators transiently increased in the bronchoalveolar lavage (BAL). Surprisingly, MDSC treatment strongly increased splenocyte numbers, however, without altering the percentage of splenic leukocyte populations. T cells of MDSC-treated TxT mice exhibited an activated phenotype characterized by expression of activation markers and elevated proliferative capacity in vitro, which was not accompanied by up-regulated exhaustion markers or unresponsiveness towards in vitro activation. Most importantly, also T cell expansion after staphylococcal enterotoxin B (SEB) stimulation in vivo was unchanged between MDSC-treated or untreated mice. After MDSC transfer, T cells preferentially exhibited a Th1 phenotype, a prerequisite to circumvent post-traumatic infectious complications. Our findings reveal a totally unexpected immunostimulatory role of adoptively transferred MDSCs in TxT and might offer options to interfere with post-traumatic malfunction of the adaptive immune response.

Burn and thoracic trauma alters fracture healing, systemic inflammation, and leukocyte kinetics in a rat model of polytrauma.

BACKGROUND: Singular traumatic insults, such as bone fracture, typically initiate an appropriate immune response necessary to restore the host to pre-insult homeostasis with limited damage to self. However, multiple concurrent insults, such as a combination of fracture, blunt force trauma, and burns (polytrauma), are clinically perceived to result in abnormal immune response leading to inadequate healing and resolution. To investigate this phenomenon, we created a model rat model of polytrauma. METHODS: To investigate relationship between polytrauma and delayed healing, we created a novel model of polytrauma in a rat which encompassed a 3-mm osteotomy, blunt chest trauma, and full-thickness scald burn. Healing outcomes were determined at 5 weeks where the degree of bone formation at the osteotomy site of polytrauma animals was compared to osteotomy only animals (OST). RESULTS: We observed significant differences in the bone volume fraction between polytrauma and OST animals indicating that polytrauma negatively effects wound healing. Polytrauma animals also displayed a significant decrease in their ability to return to pre-injury weight compared to osteotomy animals. Polytrauma animals also exhibited significantly altered gene expression in osteogenic pathways as well as the innate and adaptive immune response. Perturbed inflammation was observed in the polytrauma group compared to the osteotomy group as evidenced by significantly altered white blood cell (WBC) profiles and significantly elevated plasma high-mobility group box 1 protein (HMGB1) at 6 and 24 h post-trauma. Conversely, polytrauma animals exhibited significantly lower concentrations of plasma TNF-alpha (TNF-α) and interleukin 6 (IL-6) at 72 h post-injury compared to OST. CONCLUSIONS: Following polytrauma with burn injury, the local and systemic immune response is divergent from the immune response following a less severe singular injury (osteotomy). This altered immune response that follows was associated with a reduced capacity for wound healing.

Pharmacological inhibition of IL-6 trans-signaling improves compromised fracture healing after severe trauma.

Patients with multiple injuries frequently suffer bone fractures and are at high risk to develop fracture healing complications. Because of its key role both in systemic posttraumatic inflammation and fracture healing, the pleiotropic cytokine interleukin-6 (IL-6) may be involved in the pathomechanisms of trauma-induced compromised fracture healing. IL-6 signals are transmitted by two different mechanisms: classic signaling via the membrane-bound receptor (mIL-6R) and trans-signaling via its soluble form (sIL-6R). Herein, we investigated whether IL-6 classic and trans-signaling play different roles in bone regeneration after severe injury. Twelve-week-old C57BL/6J mice underwent combined femur osteotomy and thoracic trauma. To study the function of IL-6, either an anti-IL-6 antibody, which inhibits both IL-6 classic and trans-signaling, or a soluble glycoprotein 130 fusion protein (sgp130Fc), which selectively blocks trans-signaling, were injected 30 min and 48 h after surgery. Bone healing was assessed using cytokine analyses, flow cytometry, histology, micro-computed tomography, and biomechanical testing. Selective inhibition of IL-6 trans-signaling significantly improved the fracture healing outcome after combined injury, as confirmed by accelerated cartilage-to-bone transformation, enhanced bony bridging of the fracture gap and improved mechanical callus properties. In contrast, global IL-6 inhibition did not affect compromised fracture healing. These data suggest that classic signaling may mediate beneficial effects on bone repair after severe injury. Selective inhibition of IL-6 trans-signaling might have therapeutic potential to treat fracture healing complications in patients with concomitant injuries.

Effects of Prior Psychosocial Trauma on Subsequent Immune Response After Experimental Thorax Trauma.

Overshooting inflammation during the early phase after blunt thorax trauma promotes the development of acute respiratory distress syndrome, multiple organ failure, and subsequent mortality. Given that individuals diagnosed with stress-related disorders are characterized by chronic low-grade inflammation, we hypothesize that "psychosocial traumatic preload" poses a risk factor for the abovementioned complications after thorax trauma.Here, we used the chronic subordinate colony housing (CSC) paradigm to induce "psychosocial traumatic preload" and systemic low-grade immune activation in male mice, indicated by elevated plasma concentrations of different inflammatory mediators. Subsequent thorax trauma was induced in anaesthetized mice by a single blast wave centered on the thorax; SHAM animals were exposed to anesthesia only. Mice were killed 2, 6, and 24 h after thorax trauma or SHAM treatment.Independent of thorax trauma, CSC caused an increase in adrenal weight, and a decrease in thymus weight, indicating that the stress paradigm worked reliably. Moreover, although lung histology was not affected by prior stress, CSC exposure aggravated the early immune response after thorax trauma, indicated by elevated myeloperoxidase lung concentrations in thorax trauma-exposed CSC versus respective single-housed control (SHC) mice (2 h). Furthermore, thorax trauma caused an increase in total bronchoalveolar lavage fluid (BAL) protein (24 h), BAL C5a (2 h), BAL cell counts (24 h), and BAL keratinocyte chemoattractant (6 h and 24 h) in CSC but not SHC mice.Our data indicate that repeated psychosocial traumatization during adulthood moderately aggravates the local immune response toward thorax trauma, but overall may be considered as a rather minor risk factor in terms of thorax trauma-associated complications.

Frequency and perforin expression of different lymphocyte subpopulations in patients with lower limb fracture and thoracic injury.

INTRODUCTION: Trauma with multiple injuries is associated with a high risk of complications, which may be related to excessive stimulation of inflammatory and anti-inflammatory responses. Although the effects of polytrauma on the immune response have been well established at the cellular and molecular levels, there is little information about the changes in the cytolytic potential of immunocompetent cells, including expression of cytotoxic molecules such as perforin. Therefore, the objective of the present study was to analyse and compare differences in the frequency and perforin expression of leukocyte subpopulations in the peripheral blood of patients with lower limb fracture, thoracic injury, and simultaneous lower limb fracture and thoracic injury. PATIENTS AND METHODS: Forty-five patients with trauma injury (15 patients with lower limb injury, 15 patients with thoracic injury, and 15 patients with simultaneous lower limb and thoracic injury) were included in the study. Peripheral blood of 15 sex- and age-matched healthy volunteers served as the control group. Peripheral blood samples were taken from all subjects included in the study and peripheral blood mononuclear cells were isolated by gradient centrifugation. The frequency of T lymphocytes, natural killer (NK) and NK T cells, and their subsets, as well as their perforin expression levels were simultaneously detected and analysed by flow cytometry. RESULTS: There was a statistically significant decrease in the frequency of T lymphocytes, NK and NK T cells as well as perforin expression in the patients with simultaneous lower limb and thoracic injury compared with the other two groups, with a predominantly marked decrease in NK and NK T cells. CONCLUSION: The decrease in the frequency and cytotoxic potential of peripheral blood lymphocytes is related to the severity of trauma injury, which can explain the underlying mechanism contributing to complication occurrence.

A new multiple trauma model of the mouse.

BACKGROUND: Blunt trauma is the most frequent mechanism of injury in multiple trauma, commonly resulting from road traffic collisions or falls. Two of the most frequent injuries in patients with multiple trauma are chest trauma and extremity fracture. Several trauma mouse models combine chest trauma and head injury, but no trauma mouse model to date includes the combination of long bone fractures and chest trauma. Outcome is essentially determined by the combination of these injuries. In this study, we attempted to establish a reproducible novel multiple trauma model in mice that combines blunt trauma, major injuries and simple practicability. METHODS: Ninety-six male C57BL/6 N mice (n = 8/group) were subjected to trauma for isolated femur fracture and a combination of femur fracture and chest injury. Serum samples of mice were obtained by heart puncture at defined time points of 0 h (hour), 6 h, 12 h, 24 h, 3 d (days), and 7 d. RESULTS: A tendency toward reduced weight and temperature was observed at 24 h after chest trauma and femur fracture. Blood analyses revealed a decrease in hemoglobin during the first 24 h after trauma. Some animals were killed by heart puncture immediately after chest contusion; these animals showed the most severe lung contusion and hemorrhage. The extent of structural lung injury varied in different mice but was evident in all animals. Representative H&E-stained (Haematoxylin and Eosin-stained) paraffin lung sections of mice with multiple trauma revealed hemorrhage and an inflammatory immune response. Plasma samples of mice with chest trauma and femur fracture showed an up-regulation of IL-1ß (Interleukin-1ß), IL-6, IL-10, IL-12p70 and TNF-α (Tumor necrosis factor- α) compared with the control group. Mice with femur fracture and chest trauma showed a significant up-regulation of IL-6 compared to group with isolated femur fracture. CONCLUSIONS: The multiple trauma mouse model comprising chest trauma and femur fracture enables many analogies to clinical cases of multiple trauma in humans and demonstrates associated characteristic clinical and pathophysiological changes. This model is easy to perform, is economical and can be used for further research examining specific immunological questions.

MDSCs are induced after experimental blunt chest trauma and subsequently alter antigen-specific T cell responses.

Severe blunt chest trauma (TxT) induces a strong inflammatory response with posttraumatic immune suppression pointing to an impaired adaptive immune response. Since CD11b+Gr-1+-expressing myeloid-derived suppressor cells (MDSCs) are induced after inflammation and suppress T cell responses, MDSC induction and their impact on T cell functions was analysed in an experimental TxT model. MDSCs were induced preferentially in the lung until 24 hours after TxT. Although MDSC numbers were only faintly increased in the spleen, splenic MDSCs isolated after TxT strongly inhibited alloantigen-induced T cell proliferation in vitro. Suppressive activity correlated with increased expression of arginase-1 and iNOS. MDSCs also prevented antigen-induced T cell expansion in vivo, since staphylococcus enterotoxin B (SEB)-induced proliferation of vß8+ T cells was impaired in TxT mice in the presence of CD11b+Gr-1+ cells. Surprisingly, MDSCs were not involved in shifting T cells into Th2 cells, characterized by the secretion of cytokines impairing cell-mediated immunity and promoting immunosuppression. Instead, the presence of CD11b+Gr-1+ cells was required for efficient IL-2, IFN-γ and TNFα production after antigenic stimulation, indicating, that elevation of MDSCs early after traumatic injuries might contribute to restrict the initial inflammatory response by alleviating T cell expansion, however, without impeding Th1 functions.

Hypercapnic Conditions After Experimental Blunt Chest Trauma Increase Efferocytosis of Alveolar Macrophages and Reduce Local Inflammation.

Blunt chest trauma induces severe local and systemic inflammatory alterations and an accumulation of apoptotic polymorphonuclear granulocytes (aPMN) in the lungs, frequently followed by bacterial infection. Alveolar macrophages (AM) represent one of the main actors for their clearance. However, little is known regarding regulatory and influencing factors of AM efferocytic and phagocytic activities. In this context, we investigated the influence of impaired gas exchange on AM activity.Male rats underwent blunt chest trauma or sham procedure and aPMN or Escherichia coli (E. coli) were instilled. Subsequently, the efferocytic and phagocytic activities were assessed by analyzing AM obtained from bronchoalveolar lavage fluids at three time points. To determine whether efferocytic and phagocytic activities of AM are affected by shifting gas concentrations, AM were subjected in vitro to hypoxic and hypercapnic conditions.Trauma significantly upregulated the capacity of AM to ingest E. coli starting 24 h after trauma, whereas the aPMN uptake rate remained virtually unchanged. In vitro, AM reacted to hypercapnic conditions by enhanced efferocytosis associated with increased release of anti-inflammatory cytokines. Additionally, phagocytosis and the pro-inflammatory reaction of AM after trauma appeared to be impaired. In contrast, hypoxic conditions displayed no regulatory effect on AM.In conclusion, blunt chest trauma enhances phagocytic activity of AM. On the other hand, hypercapnic conditions in the lungs may significantly contribute to the clearance of aPMN. The application of CO2 in clinical settings must be properly assessed, with the benefits of CO2 balanced against the detrimental effects of impaired bacterial clearance.

Shed Pleural Blood from Traumatic Hemothorax Contains Elevated Levels of Pro-Inflammatory Cytokines.

PURPOSE: The autotransfusion of unwashed (or unprocessed) shed hemothorax blood (USHB) in trauma patients is widely assumed to be beneficial; however, the inflammatory potential of shed pleural blood has not been thoroughly studied. Since previous studies have documented marked changes in coagulation function of shed pleural blood, we hypothesized that its level of inflammatory cytokines would be elevated. METHODS: A prospective observational study of trauma patients in whom cytokine levels from USHB were compared to venous samples from healthy volunteers was conducted. Differences between the cytokine content of patient-derived samples were compared to those from healthy subjects. RESULTS: There was a statistically significant increase in pro-inflammatory cytokines (IL-6, IL-8, TNFα, GM-CSF), a pro-inflammatory Th-1 cytokine (IFNγ), and anti-inflammatory Th-2 cytokines (IL-4 and IL-10) in shed pleural blood over four hours when compared with samples from healthy controls (P <0.05). Cytokine levels in USHB are approximately 10- to 100-fold higher compared with healthy control venous samples. CONCLUSIONS: USHB, even collected within the accepted four-hour window, contains significantly elevated cytokine levels, suggesting the potential for deleterious effects from autotransfusion. Randomized trials are needed to determine the safety and efficacy of autotransfusion in trauma patients.

Physiological and immune-biological characterization of a long-term murine model of blunt chest trauma.

Blunt chest trauma causes pulmonary and systemic inflammation. It is still a matter of debate whether the long-term course of this inflammatory response is associated with persistent impairment of lung function. We hypothesized that an increase of inflammatory biomarkers may still be present at later time points after blunt chest trauma, eventually, despite normalized lung mechanics and gas exchange. Anesthetized spontaneously breathing male C57BL/6J mice underwent a blast wave-induced blunt chest trauma or sham procedure. Twelve and 24 h later, blood gases and lung mechanics were measured, together with blood, bronchoalveolar lavage (BAL), and tissue cytokine concentrations (multiplex cytokine kit); heme oxygenase 1 (HO-1), activated caspase-3, Bcl-xL, and Bax expression (Western blotting); nuclear factor-κB activation (electrophoretic mobility shift assay); nitrotyrosine formation; and purinergic (P2XR4 and P2XR7) receptor expression (immunohistochemistry). Histological damage was assessed by hematoxylin and eosin and periodic acid-Schiff staining. High-resolution respirometry allowed assessing mitochondrial respiration in diaphragm biopsies. Chest trauma significantly increased tissue and BAL cytokine levels, associated with a significant increase in HO-1, purinergic receptor expression, and tissue nitrotyrosine formation. In contrast, lung mechanics, gas exchange, and histological damage did not show any significant difference between sham and trauma groups. Activation of the immune response remains present at later time points after murine blunt chest trauma. Discordance of the increased local inflammatory response and preserved pulmonary function may be explained by a dissociation of the immune response and lung function, such as previously suggested after experimental sepsis.

Thorax injury lowers resistance to infection in Drosophila melanogaster.

The route of infection can profoundly affect both the progression and outcome of disease. We investigated differences in Drosophila melanogaster defense against infection after bacterial inoculation into two sites--the abdomen and the thorax. Thorax inoculation results in increased bacterial proliferation and causes high mortality within the first few days of infection. In contrast, abdomen inoculation results in minimal mortality and lower bacterial loads than thorax inoculation. Inoculation into either site causes systemic infection. Differences in mortality and bacterial load are due to injury of the thorax and can be recapitulated by abdominal inoculation coupled with aseptic wounding of the thorax. This altered resistance appears to be independent of classical immune pathways and opens new avenues of research on the role of injury during defense against infection.

Penetrating thorax injury leads to mild systemic activation of neutrophils without inflammatory complications.

INTRODUCTION: Trauma is one of the major causes of morbidity and mortality. Thoracic injuries are associated with inflammatory complications such as ARDS. The pathogenesis of this complication after pulmonary injury is incompletely understood, but neutrophils are thought to play a pivotal role. The aim of this project was to gain more insight in the role of thoracic injuries in the pathophysiological processes that link systemic neutrophil activation with inflammatory complications after trauma. METHODS: In this prospective cohort study fifty-five patients with isolated penetrating thoracic injury were included at a level one Trauma Unit. Blood samples were analysed for neutrophil phenotype with the use of flowcytometry within 3 h of trauma and repeated six and 24 h after injury. The presence of inflammatory complications (e.g. ARDS or sepsis/septic shock) was assessed during admission, and this was related to the neutrophil phenotpe. RESULTS: The clinical follow-up of fifty-three patients was uneventful. Only two patients developed an inflammatory complication. Within 3 h after trauma, neutrophils showed a decreased expression of FcγRII (p=0.007) and FcγRIII (p=0.001) compared to healthy individuals. After 6 h, expression of active FcγRII (p=0.017), C5aR (p=0.004) and CAECAM8 (p=0.043) increased, whereas L-selectin (p=0.002) decreased. After 24 h also CXCR-2 (CD182) expression increased compared to healthy individuals (p=0.001). CONCLUSIONS: Penetrating thoracic trauma leads to a distinct primed activation status of circulating neutrophils within hours. In addition to activation of cells, both young and reverse migrated neutrophils are released into the circulation. This degree of systemic inflammation does not exceed a threshold of inflammation that is needed for the development of inflammatory complications like ARDS.

The role of thoracic trauma in inflammatory responses, apoptosis and bacterial translocation following multiple traumas.

BACKGROUND: Blunt chest trauma and its complications are commonly encountered in emergency medicine. Herein, we used a rat model to investigate the role of thoracic trauma in inflammation, apoptosis and bacterial translocation following multiple traumas. METHODS: Ninety Wistar rats were divided equally into nine groups. Rats underwent a standardized blunt thoracic and/or head trauma and were sacrificed 24 or 48 hours after the trauma. Specimens from various organs and blood samples were collected and quantitatively cultured for aerobic organisms. Interleukins, TNF-α, and MCP-1 levels were assessed in the sera and markers of apoptosis were detected in the lungs. RESULTS: Levels of interleukins, TNF-α and MCP-1 in all of the groups undergoing trauma were significantly higher than those of the control group (p=0.001). Levels of apoptotic cells in the groups undergoing head and thoracic trauma (HTT) were significantly higher than those of the control group (p=0.009). Light microscopic evaluation indicated that damage in the HTT groups was significantly higher than that in the control group. The incidence of bacterial translocation was also significantly higher in the HTT groups (p=0.003). CONCLUSION: Multiple inflammatory mediators are activated in multiple traumas (including blunt thoracic trauma), which allow bacterial translocation and apoptotic processes to occur. Our results indicate that thoracic trauma plays a major role in post-traumatic bacterial translocation, inflammation, and apoptosis following multiple traumas.

Cytokine productive capacity of alveolar macrophages and Kupffer cells after femoral fracture and blunt chest trauma in a murine trauma model.

INTRODUCTION: Specific cellular and inflammatory factors that contribute to the severity of pulmonary dysfunction after blunt chest trauma and osteosynthesis of femoral fractures are yet not fully understood. Therefore, we investigated alterations of the cytokine productive capacity of alveolar macrophages (AM) and Kupffer cells (KC) after femoral fracture stabilized with intramedullary pin with or without blunt chest trauma. MATERIALS AND METHODS: In male C57BL/6N mice an intramedullary pin was implanted in an intact femur as the sham procedure. In trauma groups mice either received an isolated femoral fracture with subsequent fracture stabilization with an intramedullary pin (group Fx) or a combined trauma of blunt chest trauma and femur fracture also stabilized by an intramedullary pin (group TTFx). Animals were sacrificed 0h, 6h, 12h, 24h and 3d after trauma induction. Cytokine concentrations were measured in plasma and supernatant of cultivated AM and KC by FACS analysis. Pulmonary and hepatic infiltration of polymorphonuclear leukocytes (PMN) was determined by Ly6G-staining. RESULTS: At 6h, isolated femoral fracture with intramedullary stabilization resulted in a significantly increased productive capacity of KC (IL-6, TNF-α, CCL2, CCL3, CCL5 and CCL7) compared to sham animals. Combined trauma additionally resulted in an increased productive capacity of AM (IL-6, TNF-α, CCL2, CCL3, CCL4, CCL5 and CCL7) at 6h and the effect was prolonged up to 3d compared to controls. Combined trauma also led to a significant higher amount of plasma CCL2 at 3d and plasma CCL7 at 6h after the insult compared to group Fx. Compared to shams, pulmonary and hepatic infiltrations of PMNs were increased in group Fx and TTFx after 6h, but in the combined trauma model the effect was prolonged up to 3d. CONCLUSION: An intramedullary stabilized femur fracture alone results in a significant activation of the immune response. The combination of femoral fracture and blunt chest trauma however, results in an increased and prolonged activation of the inflammatory response. Transferred to the clinical setting, these results emphasize the critical role of severe chest trauma for treatment strategies of femoral fractures in multiple trauma patients.

C5aR-antagonist significantly reduces the deleterious effect of a blunt chest trauma on fracture healing.

Confirming clinical evidence, we recently demonstrated that a blunt chest trauma considerably impaired fracture healing in rats, possibly via the interaction of posttraumatic systemic inflammation with local healing processes, the underlying mechanisms being unknown. An important trigger of systemic inflammation is the complement system, with the potent anaphylatoxin C5a. Therefore, we investigated whether the impairment of fracture healing by a severe trauma resulted from systemically activated complement. Rats received a blunt chest trauma and a femur osteotomy stabilized with an external fixator. To inhibit the C5a-dependent posttraumatic systemic inflammation, half of the rats received a C5aR-antagonist intravenously immediately and 12 h after the thoracic trauma. Compared to the controls (control peptide), the treatment with the C5aR-antagonist led to a significantly increased flexural rigidity (three-point-bending test), an improved bony bridging of the fracture gap, and a slightly larger and qualitatively improved callus (µCT, histomorphometry) after 35 days. In conclusion, immunomodulation by a C5aR-antagonist could abolish the deleterious effects of a thoracic trauma on fracture healing, possibly by influencing the function of inflammatory and bone cells locally at the fracture site. C5a could possibly represent a target to prevent delayed bone healing in patients with severe trauma.

Alveolar macrophage phagocytosis is enhanced after blunt chest trauma and alters the posttraumatic mediator release.

Blunt chest trauma is known to induce a pulmonary invasion of short-lived polymorphonuclear neutrophils and apoptosis of alveolar epithelial type 2 (AT2) cells. Apoptotic cells are removed by alveolar macrophages (AMΦ). We hypothesized that chest trauma alters the phagocytic response of AMΦ as well as the mediator release of AMΦ during phagocytosis. To study this, male Sprague-Dawley rats were subjected to blunt chest trauma. Phagocytosis assays were performed in AMΦ isolated 2 or 24 h after trauma with apoptotic cells or opsonized beads. Phagocytosis of apoptotic AT2 cells by unstimulated AMΦ was significantly increased 2 h after trauma. At 24 h, AMΦ from traumatized animals, stimulated with phorbol-12-myristate-13-acetate, ingested significantly more apoptotic polymorphonuclear neutrophils than AMΦ from sham animals. Alveolar macrophages after trauma released significantly higher levels of tumor necrosis factor α, macrophage inflammatory protein 1α, and cytokine-induced neutrophil chemoattractant 1 when they incorporated latex beads, but significantly lower levels of interleukin 1ß and macrophage inflammatory protein 1α when they ingested apoptotic cells. In vivo, phagocytosis of intratracheally instilled latex beads was decreased in traumatized rats. The bronchoalveolar lavage concentrations of the phagocytosis-supporting surfactant proteins A and D after blunt chest trauma were slightly decreased, whereas surfactant protein D mRNA expression in AT2 cells was significantly increased after 2 h. These findings indicate that chest trauma augments the phagocytosis of apoptotic cells by AMΦ. Phagocytosis of opsonized beads enhances and ingestion of apoptotic cells downregulates the immunologic response following lung contusion. Our data emphasize the important role of phagocytosis during posttraumatic inflammation after lung contusion.