Sascha Flohé

Relation of a TNF Gene Polymorphism to Severe Sepsis in Trauma Patients

OBJECTIVE To investigate the relation of the biallelic Nco1 restriction fragment length polymorphism in the first intron of the tumor necrosis factor (TNF) beta gene with the development of severe sepsis in multiply injured patients. SUMMARY BACKGROUND DATA The biallelic Nco1 polymorphism of the TNFbeta gene has been described to be associated with autoimmune diseases and with the mortality rate in severe sepsis. Therefore, the Nco1 polymorphism may be associated with the clinical finding that despite comparable risk factors, posttraumatic sepsis develops in some patients but not others. METHODS The study group consisted of 110 patients with severe blunt trauma (Injury Severity Score > or = 17). Typing of each patient for the biallelic Nco1 polymorphism was performed by analyzing restriction fragments of an Nco1-digested DNA fragment obtained using polymerase chain reaction. Genotypes were then related to the occurrence of severe posttraumatic sepsis and TNFalpha serum concentrations. RESULTS Fifty-seven patients showed an uncomplicated posttraumatic recovery, and severe sepsis developed in 53 patients. The overall allele frequency (TNFB1 0.29, TNFB2 0.71) and genotype distribution (TNFB1 homozygous 7.3%, TNFB1/TNFB2 42.7%, TNFB2 homozygous 50%) were in agreement with the distribution in healthy volunteers. Genotype distribution in patients with an uncomplicated clinical course was significantly different from that in patients with severe posttraumatic sepsis. Development of severe posttraumatic sepsis was significantly increased in patients homozygous for the allele TNFB2. In patients with severe posttraumatic sepsis, TNFalpha serum concentrations were significantly higher in TNFB2-homozygous individuals compared with heterozygous and TNFB1 -homozygous individuals. The age- and injury-matched odds ratio for the homozygous TNFB2 genotype compared with the heterozygous genotype was 5.22 (p = 0.007, 95% confidence interval 1.6 to 17.9). CONCLUSIONS In multiply injured patients, the Nco1 polymorphism within the TNFbeta gene is associated with the development of severe posttraumatic sepsis and with increased TNFalpha serum levels when severe sepsis has occurred. This suggests a genetic determination of the individual inflammatory response after infection or tissue damage, which significantly influences susceptibility to severe nosocomial infections.

Human heat shock protein 60 induces maturation of dendritic cells versus a Th1-promoting phenotype.

Heat shock protein (HSP) 60 nonspecifically activates cells of the innate immune system. In the present study, we characterized the effects of human HSP60 maturation, cytokine release, and T cell-activating capacity of bone marrow-derived dendritic cells (DC). Furthermore, we analyzed HSP60-induced signal transduction in DC. HSP60 strongly stimulated DC for maturation and release of TNF-α, IL-12, and IL-1β. However, HSP60 elicited only a weak IL-10 response in DC suggesting a Th1 bias. HSP60-treated DC induced proliferation of allogeneic T cells. Again, a Th1 bias was noted in that cocultures of allogeneic T cells and HSP60-treated DC released IFN-γ but only small amounts of IL-10 and no detectable IL-4. Signaling via Toll-like receptor 4 was involved in HSP60-induced cytokine release and maturation because DC of C3H/HeJ mice with a mutant Toll-like receptor 4 showed deficient response to HSP60. HSP60 was found to rapidly activate the mitogen-activated protein kinases p38, c-Jun N-terminal kinase, and extracellular signal-regulated kinase as well as IκB in DC. Phosphorylation of these signaling molecules was also mediated by LPS, but with much slower kinetics. Thus, HSP60 stimulates DC more rapidly than LPS and elicits a Th1-promoting phenotype. These results suggest that DC play a pivotal role in priming for destructive Th1-type responses at sites of local HSP60 release.

Antigen-pulsed epidermal Langerhans cells protect susceptible mice from infection with the intracellular parasite Leishmania major.

Efficient vaccination against the parasite Leishmania major, the causative agent of human cutaneous leishmaniasis, requires the development of a resistance‐promoting CD4+ ‐mediated Th1 response. Epidermal Langerhans cells (LC) are critically involved in the induction of the primary immune response to Leishmania infection. They are able to ingest the parasites, to express MHC class II molecules with extraordinarily long half‐life and to activate naive L. major ‐specific Th cells. Considering these unique properties, we studied the capacity of LC to mediate resistance to L. major in vivo. A single i.v. application of LC that had been pulsed with L. major antigen in vitro induced the protection in susceptible BALB/c mice against subsequent challenges with L. major parasites. Resistance could neither be induced by unpulsed LC, nor by L. major antigen alone or by L. major ‐pulsed macrophages. Development of resistance was paralleled by a reduced parasite burden and by a shift of the cytokine expression towards a Th1‐like pattern. In contrast, control mice developed a Th2 response. In vitro exposure of LC to L. major antigen induced the expression of IL‐12 (p40) mRNA. In conclusion, our data demonstrate that LC are able to serve as a natural adjuvant and to induce a protective immune response to L. major infection. This effect is based on the initiation of a Th1‐like response that is likely to be mediated by IL‐12.

Depletion of neutrophil extracellular traps in vivo results in hypersusceptibility to polymicrobial sepsis in mice

IntroductionAlthough the formation of neutrophil (PMN) extracellular traps (NETs) has been detected during infection and sepsis, their role in vivo is still unclear. This study was performed in order to evaluate the influence of NETs depletion by administration of recombinant human (rh)DNase on bacterial spreading, PMN tissue infiltration and inflammatory response in a mouse model of polymicrobial sepsis.MethodsIn a prospective controlled double-armed animal trial, polymicrobial sepsis was induced by cecal ligation and puncture (CLP). After CLP, mice were treated with rhDNase or phosphate buffered saline, respectively. Survival, colony forming unit (CFU) counts in the peritoneal cavity, lung, liver and blood were determined. PMN and platelet counts, IL-6 and circulating free (cf)-DNA/NETs levels were monitored. PMN infiltration, as well as organ damage, was analyzed histologically in the lungs and liver. Capability and capacity of PMN to form NETs were determined over time.Resultscf-DNA/NETs were found to be significantly increased 6, 24, and 48 hours after CLP when compared to the levels determined in sham and naïve mice. Peak levels after 24 hours were correlated to enhanced capacity of bone marrow-derived PMN to form NETs after ex vivo stimulation with phorbol-12-myristate-13-acetate at the same time. rhDNase treatment of mice resulted in a significant reduction of cf-DNA/NETs levels 24 hours after CLP (P < 0.001). Although overall survival was not affected by rhDNase treatment, median survival after 24 hours was significantly lower when compared with the CLP group (P < 0.01). In mice receiving rhDNase treatment, CFU counts in the lung (P < 0.001) and peritoneal cavity (P < 0.05), as well as serum IL-6 levels (P < 0.001), were found to be already increased six hours after CLP. Additionally, enhanced PMN infiltration and tissue damage in the lungs and liver were found after 24 hours. In contrast, CFU counts in mice without rhDNase treatment increased later but more strongly 24 hours after CLP (P < 0.001). Similarly, serum IL-6 levels peaked after 24 hours (P < 0.01).ConclusionsThis study shows, for the first time, that depletion of NETs by rhDNase administration impedes the early immune response and aggravates the pathology that follows polymicrobial sepsis in vivo.

Influence of surgical intervention in the immune response of severely injured patients

Objective Primary events such as severe injury and elective surgery cause a deterioration of the immune response measurable by reduction of expression of HLA-DR on monocytes or ex vivo LPS-induced TNFα production. The further influence of secondary surgery after severe injury on the immune response remains unresolved.Design Prospective observation study.Setting Surgical intensive care unit of an university hospital.Patients Sixteen severely injured patients with an ISS >25 points.Measurements and results On day 1 after trauma and immediately before secondary surgery, mean fluorescence intensity (MFI) of HLA-DR expression on monocytes and TNFα ex vivo synthesis was significantly reduced compared to healthy donors. Overall, surgical intervention during the second week after trauma caused no further reduction of HLA-DR expression on monocytes and of the ex vivo TNFα-synthesis. However, major surgery such as intramedullary nailing or pelvic osteosynthesis caused reduction of the HLA-DR expression and TNFα-synthesis, whereas, minor surgical interventions such as osteosynthesis on peripheral joints exhibited no significant effects on the immune response. Surgical intervention performed to clear septic foci normalised immune response by elevating HLA-DR expression on monocytes and ex vivo TNFα synthesis. Severe injury caused elevated serum IL-10 levels, whereas secondary surgery did not induce a further increase in serum IL-10 levels.Conclusion This study shows that initial trauma as well as major secondary surgery causes a suppression of immune functions, whereas minor secondary surgery does not cause significant immune disturbance.

Invited review: Deterioration of the immune system after trauma: signals and cellular mechanisms

Multiple trauma leads to a deterioration of the immune system. On the one hand, hyperinflammation mediates remote organ damage and may lead to multi-organ failure. On the other hand, immunosuppression develops and promotes an enhanced risk to acquire infectious complications after trauma. The mechanisms that underlie these opposing consequences of trauma are not yet completely understood. There is increasing evidence that endogenous danger signals that derive from destroyed tissues play a role in trauma-induced immune dysfunction. Here, we give an overview on the common animal models that are used to investigate trauma-induced pathology, potential signals and cellular mechanisms that support the imbalance between inflammation and counter-regulation after trauma.

Effect of granulocyte-macrophage colony-stimulating factor on the immune response of circulating monocytes after severe trauma.

ObjectiveSevere injury compromises functions of the antigen-presenting immune cells, resulting in an increased vulnerability toward bacterial sepsis. Support of the immune capabilities contributes a desirable therapeutic option in high-risk patients. Factors possessing immunostimulating properties such as granulocyte-macrophage colony-stimulating factor (GM-CSF) may serve as potential tools to compensate immunosuppression caused by severe trauma. In the present study, therefore, GM-CSF was examined with regard to its capacity to overcome trauma-induced down-regulation of immune functions. DesignProspective clinical experimental study. SettingUniversity hospital intensive care unit and research facility. PatientsSeverely injured patients with >25 points on the Injury Severity Score. InterventionsBlood samples of severely injured patients were incubated in vitro with 10 ng/mL GM-CSF for 6 hrs. MeasurementsHuman leukocyte antigen (HLA)-DR expression on monocytes was analyzed by flow cytometry, lipopolysaccharide-induced tumor necrosis factor (TNF)&agr; and interleukin-10 production of blood samples was measured by means of enzyme-linked immunoabsorbent assay. Main ResultsCompared with blood specimens of healthy donors, ex vivo endotoxin-induced TNF&agr; production and HLA-DR expression on monocytes were significantly reduced in blood of trauma patients. Ex vivo treatment of blood specimens with GM-CSF increased HLA-DR expression and TNF&agr; production stimulated by lipopolysaccharides in both healthy volunteers and patients on day 1 after trauma. Blood samples of patients with an uneventful recovery showed nearly normal TNF&agr; synthesis and HLA-DR expression after 2–3 wks, whereas TNF&agr; production and HLA-DR expression of patients with sepsis and multiple organ failure remained at low levels. In the sepsis/multiple organ failure group, GM-CSF also enhanced HLA-DR expression and TNF&agr; production, although the levels of the volunteers’ blood were not reached. ConclusionsThe presented data show that trauma- and sepsis-induced depression of monocyte functions can be counteracted by GM-CSF in vitro, suggesting that this substance may serve as support of immune functions in severely injured patients.

Origin of immunomodulation after soft tissue trauma: potential involvement of extracellular heat-shock proteins.

Severe injury may lead to immunosuppression, multiple organ failure, and death. The aim of the study was to investigate the direct impact of soft tissue destruction on the development of trauma-associated immunomodulation. Hip surgery was considered to represent an isolated soft tissue trauma that allowed for the examination of changes taking place locally at the site of trauma or systemically with regard to monocyte function and leukocyte redistribution. Peripheral blood and wound fluid collected from the drains of 21 patients after hip surgery were analyzed to determine the cellular composition and/or the responsiveness of mononuclear cells (MNCs) to lipopolysaccharide (LPS). Different factors present in the wound fluids were tested for their capacity to modulate the MNC of healthy individuals with regard to cytokine and chemokine secretion. We found that various factors, including heat-shock protein (HSP) 60 and HSP70, were locally released at the site of soft tissue trauma and could be detected in wound fluids. The wound fluid-derived MNC (but not the peripheral blood-derived MNC) showed an impaired capacity to release TNF-&agr; after LPS stimulation. Cell-free wound fluid suppressed in healthy individuals the LPS-induced TNF-&agr; secretion by MNC. After surgery, granulocytosis was found in peripheral blood and in wound fluids, but monocytopenia was restricted to wound fluids. In parallel, wound fluids induced in healthy individuals the release by MNC of distinct chemokines specific for granulocytes and monocytes. These wound fluid-mediated effects of TNF-&agr; suppression and chemokine induction could be mimicked by recombinant human HSP70 and, in part, by HSP60. Thus, tissue-derived factors, such as HSP70 released after injury, suppress monocyte function and, therefore, might favor the development of immunosuppression after severe injury.ABBREVIATIONS-IL-interleukin; LPS-lipopolysaccharide; TNF-tumor-necrosis factor; MCP-1-monocytes-chemoattractant protein; RANTES-regulated on activation normal T expressed and secreted; HSP-heat-shock protein; MNC-mononuclear cells; PB-MNC-peripheral blood-derived MNC; WF-MNC-wound fluid-derived MNC

Diversity of interferon gamma and granulocyte-macrophage colony-stimulating factor in restoring immune dysfunction of dendritic cells and macrophages during polymicrobial sepsis.

The development of immunosuppression during polymicrobial sepsis is associated with the failure of dendritic cells (DC) to promote the polarization of T helper (Th) cells toward a protective Th1 type. The aim of the study was to test potential immunomodulatory approaches to restore the capacity of splenic DC to secrete interleukin (IL) 12 that represents the key cytokine in Th1 cell polarization. Murine polymicrobial sepsis was induced by cecal ligation and puncture (CLP). Splenic DC were isolated at different time points after CLP or sham operation, and stimulated with bacterial components in the presence or absence of neutralizing anti-IL-10 antibodies, murine interferon (IFN) γ, and/or granulocyte macrophage colony-stimulating factor (GM-CSF). DC from septic mice showed an impaired capacity to release the pro-inflammatory and Th1-promoting cytokines tumor necrosis factor α, IFN-γ, and IL-12 in response to bacterial stimuli, but secreted IL-10. Endogenous IL-10 was not responsible for the impaired IL-12 secretion. Up to 6 h after CLP, the combined treatment of DC from septic mice with IFN-γ and GM-CSF increased the secretion of IL-12. Later, DC from septic mice responded to IFN-γ and GM-CSF with increased expression of the co-stimulatory molecule CD86, while IL-12 secretion was no more enhanced. In contrast, splenic macrophages from septic mice during late sepsis responded to GM-CSF with increased cytokine release. Thus, therapy of sepsis with IFN-γ/GM-CSF might be sufficient to restore the activity of macrophages, but fails to restore DC function adequate for the development of a protective Th1-like immune response.

Molecular Mechanisms Underlying Delayed Apoptosis in Neutrophils from Multiple Trauma Patients with and without Sepsis

Delayed neutrophil apoptosis and overshooting neutrophil activity contribute to organ dysfunction and subsequent organ failure in sepsis. Here, we investigated apoptotic signaling pathways that are involved in the inhibition of spontaneous apoptosis in neutrophils isolated from major trauma patients with uneventful outcome as well as in those with sepsis development. DNA fragmentation in peripheral blood neutrophils showed an inverse correlation with the organ dysfunction at d 10 after trauma in all patients, supporting the important role of neutrophil apoptosis regulation for patient’s outcome. The expression of the antiapoptotic Bcl-2 protein members A1 and Mcl-1 were found to be diminished in the septic patients at d 5 and d 10 after trauma. This decrease was also linked to an impaired intrinsic apoptosis resistance, which has been previously shown to occur in neutrophils during systemic inflammation. In patients with sepsis development, delayed neutrophil apoptosis was found to be associated with a disturbed extrinsic pathway, as demonstrated by reduced caspase-8 activity and Bid truncation. Notably, the expression of Dad1 protein, which is involved in protein N-glycosylation, was significantly increased in septic patients at d 10 after trauma. Taken together, our data demonstrate that neutrophil apoptosis is regulated by both the intrinsic and extrinsic pathway, depending on patient’s outcome. These findings might provide a molecular basis for new strategies targeting cell death pathways in apoptosis-resistant neutrophils during systemic inflammation.