Hemorrhagic Shock Induces a Rapid Transcriptomic Shift of the Immune Balance in Leukocytes after Experimental Multiple Injury.

The immune response following trauma represents a major driving force of organ dysfunction and poor outcome. Therefore, we investigated the influence of an additional hemorrhagic shock (HS) on the early posttraumatic immune dysbalance in the whole population of blood leukocytes. A well-established murine polytrauma (PT) model with or without an additional pressure-controlled HS (mean arterial pressure of 30 mmHg (±5 mmHg) for 60 mins, afterwards fluid resuscitation with balanced electrolyte solution four times the volume of blood drawn) was used. C57BL/6 mice were randomized into a control, PT, and PT + HS group with three animals in each group. Four hours after trauma, corresponding to three hours after induction of hemorrhage, RNA was isolated from all peripheral blood leukocytes, and a microarray analysis was performed. Enrichment analysis was conducted on selected genes strongly modulated by the HS. After additional HS in PT mice, the gene expression of pathways related to the innate immunity, such as IL-6 production, neutrophil chemotaxis, cell adhesion, and toll-like receptor signaling was upregulated, whereas pathways of the adaptive immune system, such as B- and T-cell activation as well as the MHC class II protein complex, were downregulated. These results demonstrate that an additional HS plays an important role in the immune dysregulation early after PT by shifting the balance to increased innate and reduced adaptive immune responses.

Role of the C5a-C5a receptor axis in the inflammatory responses of the lungs after experimental polytrauma and hemorrhagic shock.

Singular blockade of C5a in experimental models of sepsis is known to confer protection by rescuing lethality and decreasing pro-inflammatory responses. However, the role of inhibiting C5a has not been evaluated in the context of sterile systemic inflammatory responses, like polytrauma and hemorrhagic shock (PT + HS). In our presented study, a novel and highly specific C5a L-aptamer, NoxD21, was used to block C5a activity in an experimental murine model of PT + HS. The aim of the study was to assess early modulation of inflammatory responses and lung damage 4 h after PT + HS induction. NoxD21-treated PT + HS mice displayed greater polymorphonuclear cell recruitment in the lung, increased pro-inflammatory cytokine levels in the bronchoalveolar lavage fluids (BALF) and reduced myeloperoxidase levels within the lung tissue. An in vitro model of the alveolar-capillary barrier was established to confirm these in vivo observations. Treatment with a polytrauma cocktail induced barrier damage only after 16 h, and NoxD21 treatment in vitro did not rescue this effect. Furthermore, to test the exact role of both the cognate receptors of C5a (C5aR1 and C5aR2), experimental PT + HS was induced in C5aR1 knockout (C5aR1 KO) and C5aR2 KO mice. Following 4 h of PT + HS, C5aR2 KO mice had significantly reduced IL-6 and IL-17 levels in the BALF without significant lung damage, and both, C5aR1 KO and C5aR2 KO PT + HS animals displayed reduced MPO levels within the lungs. In conclusion, the C5aR2 could be a putative driver of early local inflammatory responses in the lung after PT + HS.

Remote Intestinal Injury Early After Experimental Polytrauma and Hemorrhagic Shock.

Dysfunction of the gut-blood barrier plays an important role in many diseases, such as inflammatory bowel disease, hemorrhagic shock (HS), or burn injury. However, little is known about gut barrier dysfunction after hemodynamically instable polytrauma (PT). Therefore, we aimed to evaluate the effects of PT and HS on remote intestinal damage and barrier dysfunction, especially regarding the role of zonula occludens protein 1 (ZO-1) as an important tight junction protein.Male C57BL/6 mice were subjected to either PT (thorax trauma, closed head injury, soft tissue injury, and distal femoral fracture), 60 min of pressure-controlled HS (30 ±â€Š5 mmHg), or PT+HS, or sham procedures.Animals of all trauma groups showed an increase in abdominal girth and dilation of the intestine during the experimental period, which was largest in the PT+HS group. Increased blood-tissue permeability to albumin (assessed by Evans blue dye) was found in the HS group. Experimental groups showed a slight increase in plasma concentration of intestinal fatty acid binding protein and some intestinal damage was histologically detectable. Of note, PT+HS animals revealed significantly reduced expression of ZO-1 in intestinal epithelial cells. In an in-vitro model, stimulation of human colon epithelial cells with peptidoglycan, but not with lipopolysaccharide, resulted in elevated secretion of pro-inflammatory cytokines, reflecting inflammatory activity of the intestinal epithelium.Taken together, PT and HS lead to increased permeability of the gut-blood barrier. Bacterial components may lead to production of inflammatory and chemotactic mediators by gut epithelial cells, underlining the role of the gut as an immunologically active organ.

Inflammatory and apoptotic alterations in serum and injured tissue after experimental polytrauma in mice: distinct early response compared with single trauma or "double-hit" injury.

BACKGROUND: The exact alterations of the immune system after polytrauma leading to sepsis and multiple-organ failure are poorly understood. Thus, the early local and systemic inflammatory and apoptotic response was characterized in a new polytrauma model and compared with the alterations seen after single or combined injuries. METHODS: Anesthetized C57BL/6 mice were subjected to either blunt bilateral chest trauma (Tx), closed head injury, right femur fracture including contralateral soft tissue injury, or a combination of injuries (PTx). After 2 hours or 6 hours, animals were sacrificed, and the systemic as well as the local pulmonary immune response (bronchoalveolar lavage [BAL]/plasma cytokines, lung myeloperoxidase [MPO] activity, and alveolocapillary barrier dysfunction) were evaluated along with lung/brain apoptosis (lung caspase 3 Western blotting, immunohistochemistry, and polymorphonuclear leukocytes [PMN] Annexin V). RESULTS: Hemoglobin, PO2 saturation, and pH did not differ between the experimental groups. Local BAL cytokines/chemokines were significantly increased in almost all groups, which included Tx. There was no further enhancement of this local inflammatory response in the lungs in case of PTx. At 2 hours, all groups except sham and closed head injury alone revealed an increased activity of lung MPO. However, 6 hours after injury, lung MPO remained increased only in the PTx group. Increased BAL protein levels were found, reflecting enhanced lung leakage in all groups with Tx 6 hours after trauma. Only after PTx was neutrophil apoptosis significantly decreased, whereas lung caspase 3 and plasma interleukin 6/keratinocyte chemoattractant (KC) were substantially increased. CONCLUSION: The combination of different injuries leads to an earlier systemic inflammatory response when compared with the single insults. Interestingly, only after PTx but not after single or double hits was lung apoptosis increased, and PMN apoptosis was decreased along with a prolonged presence of neutrophils in the lungs, which may therefore represent a possible pathomechanism for lung injury after polytrauma.

Silencing of fas, fas-associated via death domain, or caspase 3 differentially affects lung inflammation, apoptosis, and development of trauma-induced septic acute lung injury.

Activation of Fas signaling is a potentially important pathophysiological mechanism in the development of septic acute lung injury (ALI). However, so far the optimal targets within this signaling cascade remain elusive. Thus, we tested the hypothesis that in vivo gene silencing of Fas, Fas-associated via death domain (FADD), or caspase 3 by intratracheal administration of small interfering RNA would ameliorate ALI in a clinically relevant double-hit mouse model of trauma induced septic lung injury. Male C57Bl/6 mice received small interfering (Fas, FADD, caspase 3) or control RNA 24 h before and 12 h after blunt chest trauma or sham procedures. Polymicrobial sepsis was induced by cecal ligation and puncture 24 h after chest trauma. Twelve or 24 h later, lung tissue, plasma, and bronchoalveolar lavage fluid were harvested. During ALI, lung apoptosis (active caspase 3 Western blotting, TUNEL staining) was substantially increased when compared with sham. Silencing of caspase 3 or FADD both markedly reduced pulmonary apoptosis. Fas- and FADD-small interfering RNA administration substantially decreased lung cytokine concentration, whereas caspase 3 silencing did not reduce lung inflammation. In addition, Fas silencing markedly decreased lung neutrophil infiltration. Interestingly, only in response to caspase 3 silencing, ALI-induced lung epithelial barrier dysfunction was substantially improved, and histological appearance was beneficially affected. Taken together, downstream inhibition of lung apoptosis via caspase 3 silencing proved to be superior in mitigating ALI when compared with upstream inhibition of apoptosis via Fas or FADD silencing, even in the presence of additional anti-inflammatory effects. This indicates a major pathophysiological role of lung apoptosis and suggests the importance of other than Fas-driven apoptotic pathways in trauma-induced septic ALI.