Heike Unnewehr, Daniel Rittirsch, J Vidya Sarma, Firas Zetoune, Michael A Flierl, Mario Perl, Stephanie Denk, Manfred Weiss, Marion E Schneider, Peter N Monk, Thomas Neff, Michael Mihlan, Holger Barth, Florian Gebhard, Peter A Ward, Markus Huber-Lang[show abstract] [hide abstract]
ABSTRACT: During experimental sepsis, excessive generation of the anaphylatoxin C5a results in reduction of the C5a receptor (C5aR) on neutrophils. These events have been shown to result in impaired innate immunity. However, the regulation and fate of C5aR on neutrophils during sepsis are largely unknown. In contrast to 30 healthy volunteers, 60 patients in septic shock presented evidence of complement activation with significantly increased serum levels of C3a, C5a, and C5b-9. In the septic shock group, the corresponding decrease in complement hemolytic activity distinguished survivors from nonsurvivors. Neutrophils from patients in septic shock exhibited decreased C5aR expression, which inversely correlated with serum concentrations of C-reactive protein (CRP) and clinical outcome. In vitro exposure of normal neutrophils to native pentameric CRP led to a dose- and time-dependent loss of C5aR expression on neutrophils, whereas the monomeric form of CRP, as well as various other inflammatory mediators, failed to significantly alter C5aR levels on neutrophils. A circulating form of C5aR (cC5aR) was detected in serum by immunoblotting and a flow-based capture assay, suggestive of an intact C5aR molecule. Levels of cC5aR were significantly enhanced during septic shock, with serum levels directly correlating with lethality. The data suggest that septic shock in humans is associated with extensive complement activation, CRP-dependent loss of C5aR on neutrophils, and appearance of cC5aR in serum, which correlated with a poor outcome. Therefore, cC5aR may represent a new sepsis marker to be considered in tailoring individualized immune-modulating therapy.The Journal of Immunology 03/2013; · 5.79 Impact Factor
Article: Apoptotic and inflammatory signaling via Fas and tumor necrosis factor receptor I contribute to the development of chest trauma-induced septic acute lung injury.Sebastian Weckbach, Christoph Hohmann, Stephanie Denk, Philipp Kellermann, Markus S Huber-Lang, Bernd Baumann, Thomas Wirth, Florian Gebhard, Max Bachem, Mario Perl[show abstract] [hide abstract]
ABSTRACT: Direct acute lung injury (ALI) is still associated with a high mortality, whereas the underlying pathomechanisms are not yet fully understood. In this regard, epithelial cell death in the lungs has been attributed an important role in the pathogenesis of this clinical entity. Based on this background here, we hypothesized that signaling through Fas and tumor necrosis factor receptor 1 (TNFR-1) is involved in mediating apoptosis and inflammation in chest trauma induced septic ALI. Male C57BL/6 mice (wild-type [WT]), male mutant mice expressing nonfunctional Fas receptor (B6.MRL-Faslpr/J [lpr]) (lpr) and male TNFR-1-deficient mice (TNFR-1) were subjected to a model of direct ALI consisting of blunt chest trauma followed by cecal ligation and puncture.Cytokine/chemokine concentrations of plasma, bronchoalveolar lavage (BAL) fluids, and lung tissue were investigated as well as BAL protein and lung myeloperoxidase. Lung histology was assessed; lung caspase 3, TUNEL-positive cells, and apoptotic polymorphonuclear neutrophil were measured, followed by a survival study. Cytokine/chemokine levels in plasma, BAL, and lung tissue were markedly increased in WT animals following ALI, whereas lpr and TNFR-1 showed significantly decreased levels. BAL protein levels were substantially elevated following ALI, but lpr animals presented markedly diminished protein levels compared with WT and TNFR-1animals. Lung myeloperoxidase level was only increased 12 hours after ALI in WT animals, whereas lung myeloperoxidase levels in lpr and TNFR-1 animals were not increased compared with sham. Lung histology revealed beneficial effects in lpr and TNFR-1. Lung active caspase 3 after ALI was substantially decreased in lpr and TNFR-1 mice compared with WT. Interestingly, an early but not persisting survival benefit was observed in lpr and TNFR-1 animals. Pathomechanistically, Fas and TNFR-1 signaling contributed to the apoptotic and inflammatory response in a clinically relevant double-hit model of trauma-induced septic ALI. Moreover, this was associated with a temporary survival benefit.The journal of trauma and acute care surgery. 03/2013; 74(3):792-800.
Article: 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.Sebastian Weckbach, Christoph Hohmann, Sonja Braumueller, Stephanie Denk, Bettina Klohs, Philip F Stahel, Florian Gebhard, Markus S Huber-Lang, Mario Perl[show abstract] [hide abstract]
ABSTRACT: 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. 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). 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. 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.The journal of trauma and acute care surgery. 02/2013; 74(2):489-98.
Article: 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.Mirko Philipp Messer, Philipp Kellermann, Sascha Jörn Weber, Christoph Hohmann, Stephanie Denk, Bettina Klohs, Anke Schultze, Sonja Braumüller, Markus Stefan Huber-Lang, Mario Perl[show abstract] [hide abstract]
ABSTRACT: 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.Shock (Augusta, Ga.) 01/2013; 39(1):19-27. · 2.87 Impact Factor
Article: The role and source of tumor necrosis factor-α in hemorrhage-induced priming for septic lung injury.[show abstract] [hide abstract]
ABSTRACT: Tumor necrosis factor α (TNF-α) has been reported to be a key component of the functional priming, of both myeloid and nonmyeloid cells, that is thought to contribute to the lung's increased susceptibility to injury following shock. Not surprisingly, we found that mice deficient in TNF-α exhibited reduced acute lung injury (ALI) resultant from the combined insults of hemorrhagic shock and sepsis. However, we found that when we adoptively transferred neutrophils from mice expressing TNF-α to neutrophil-depleted mice that lacked TNF-α, they were not able to serve as priming stimulus for the development of ALI. Based on these findings, we proposed that resident lung tissue cells mediate TNF-α priming. To begin to unravel the complex signaling pathway of various resident lung tissue cells in TNF-α-induced priming, we compared the effect of local (intratracheal [i.t.]) versus systemic [intravenous (i.v.)] delivery of TNF-α small interference (siRNA). We hypothesized that alternately suppressing expression of TNF-α in lung endothelial (i.v.) or epithelial (i.t.) cells would produce a differential effect in shock-induced ALI. We found that when in vivo siRNA i.t. or i.v. against TNF-α was administered to C57/BL6 mice at 2 h after hemorrhage, 24 h before septic challenge, that systemic/i.v., but not i.t., delivery of TNF-α siRNA following hemorrhage priming significantly reduces expression of indices of ALI compared with controls. These findings suggest that an absence of local lung tissue TNF-α significantly reduces lung tissue injury following hemorrhage priming for ALI and that pulmonary endothelial and/or other possible vascular resident cells, not epithelial cells, play a greater role in mediating the TNF-α priming response in a mouse model of hemorrhage/sepsis-induced ALI.Shock (Augusta, Ga.) 05/2012; 37(6):611-20. · 2.87 Impact Factor