Adnana Paunel-Görgülü

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.

Whole Body UVA Irradiation Lowers Systemic Blood Pressure by Release of Nitric Oxide From Intracutaneous Photolabile Nitric Oxide Derivates

Rationale: Human skin contains photolabile nitric oxide derivates like nitrite and S-nitroso thiols, which after UVA irradiation, decompose and lead to the formation of vasoactive NO. Objective: Here, we investigated whether whole body UVA irradiation influences the blood pressure of healthy volunteers because of cutaneous nonenzymatic NO formation. Methods and Results: As detected by chemoluminescence detection or by electron paramagnetic resonance spectroscopy in vitro with human skin specimens, UVA illumination (25 J/cm2) significantly increased the intradermal levels of free NO. In addition, UVA enhanced dermal S-nitrosothiols 2.3-fold, and the subfraction of dermal S-nitrosoalbumin 2.9-fold. In vivo, in healthy volunteers creamed with a skin cream containing isotopically labeled 15N-nitrite, whole body UVA irradiation (20 J/cm2) induced significant levels of 15N-labeled S-nitrosothiols in the blood plasma of light exposed subjects, as detected by cavity leak out spectroscopy. Furthermore, whole body UVA irradiation caused a rapid, significant decrease, lasting up to 60 minutes, in systolic and diastolic blood pressure of healthy volunteers by 11±2% at 30 minutes after UVA exposure. The decrease in blood pressure strongly correlated (R2=0.74) with enhanced plasma concentration of nitrosated species, as detected by a chemiluminescence assay, with increased forearm blood flow (+26±7%), with increased flow mediated vasodilation of the brachial artery (+68±22%), and with decreased forearm vascular resistance (−28±7%). Conclusions: UVA irradiation of human skin caused a significant drop in blood pressure even at moderate UVA doses. The effects were attributed to UVA induced release of NO from cutaneous photolabile NO derivates.

Diagnostic accuracy of neutrophil‐derived circulating free DNA (cf‐DNA/NETs) for septic arthritis

The release of “neutrophil extracellular traps” (NETs) has been identified as a novel immune response in innate immunity. NETs are composed of neutrophil‐derived circulating free DNA (cf‐DNA) and neutrophil cytoplasm‐derived proteins such as proteases. In this study, we analyzed the putative diagnostic value of synovial cf‐DNA/NETs for identification of septic arthritis. Forty‐two patients with a joint effusion who had undergone arthrocentesis were included. From synovial fluid, cf‐DNA/NETs (j‐cf‐DNA) levels were directly quantified. Diagnostic value of j‐cf‐DNA was compared with white blood cells (WBC), synovial white blood cells (j‐WBC), C‐reactive protein (CRP), j‐IL‐6, j‐TNF alpha, j‐IL‐1 beta, and myeloperoxidase (j‐MPO). Sensitivity, specificity, positive and negative predictive value, as well as ROC‐curves for each parameter were calculated. Synovial fluid cf‐DNA/NETs values from patients with septic arthritis (3,286 ± 386 ng/ml, n = 9) were significantly increased compared to patients with noninfectious joint inflammation (1,040 ± 208 ng/ml, n = 17) or osteoarthritis (278 ± 34 ng/ml, n = 16, p < 0.01). In conjunction with j‐cf‐DNA, j‐IL‐6 and j‐IL‐1 beta were significantly elevated (p < 0.01), but WBC, CRP, and j‐WBC were not. At a cut‐off of 300 ng/ml, j‐cf‐DNA had a sensitivity of 0.89, a specificity of 1.0, a positive predictive value of 1.0, and a negative predictive value of 0.97. Receiver operation curves revealed largest areas under the curve for cf‐DNA/NETs (0.933) and j‐IL‐6 (0.951). cf‐DNA/NETs seem to be a valuable additional marker for the diagnosis of septic arthritis or periprosthetic infections. However, this result should be confirmed in a large clinical trial.

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.

Deoxyribonuclease Is a Potential Counter Regulator of Aberrant Neutrophil Extracellular Traps Formation after Major Trauma

Introduction. Neutrophil extracellular traps (NET) consist of a DNA scaffold that can be destroyed by Deoxyribonuclease (DNase). Thus DNases are potential prerequisites for natural counter regulation of NETs formation. In the present study, we determined the relationship of NETs and DNase after major trauma. Methods. Thirty-nine major trauma patients, 14 with and 25 without sepsis development were enrolled in this prospective study. Levels of cell-free (cf)-DNA/NETs and DNase were quantified daily from admission until day 9 after admission. Results. Levels of cf-DNA/NETs in patients who developed sepsis were significantly increased after trauma. In the early septic phase, DNase values in septic patients were significantly increased compared to patients without sepsis (P < 0.05). cf-DNA/NETs values correlated to values of DNase in all trauma patients and patients with uneventful recovery (P < 0.01) but not in septic patients. Recombinant DNase efficiently degraded NETs released by stimulated neutrophils in a concentration-dependent manner in vitro. Conclusions. DNase degrades NETs in a concentration-dependent manner and therefore could have a potential regulatory effect on NET formation in neutrophils. This may inhibit the antibacterial effects of NETs or protect the tissue from autodestruction in inadequate NETs release in septic patients.

Mcl-1-Mediated Impairment of the Intrinsic Apoptosis Pathway in Circulating Neutrophils from Critically Ill Patients Can Be Overcome by Fas Stimulation

The systemic inflammatory response syndrome and subsequent organ failure are mainly driven by activated neutrophils with prolonged life span, which is believed to be due to apoptosis resistance. However, detailed underlying mechanisms leading to neutrophil apoptosis resistance are largely unknown, and possible therapeutic options to overcome this resistance do not exist. Here we report that activated neutrophils from severely injured patients exhibit cell death resistance due to impaired activation of the intrinsic apoptosis pathway, as evidenced by limited staurosporine-induced mitochondrial membrane depolarization and decreased caspase-9 activity. Moreover, we found that these neutrophils express high levels of antiapoptotic Mcl-1 and low levels of proapoptotic Bax protein. Mcl-1 up-regulation was dependent on elevated concentrations of GM-CSF in patient serum. Accordingly, increased Mcl-1 protein stability and GM-CSF serum concentrations were shown to correlate with staurosporine-induced apoptosis resistance. However, cross-linking of neutrophil Fas by immobilized agonistic anti-Fas IgM resulted in caspase-dependent mitochondrial membrane depolarization and apoptosis induction. In conclusion, the observed impairment of the intrinsic pathway and the resulting apoptosis resistance may be overcome by immobilized agonistic anti-Fas IgM. Targeting of neutrophil Fas by immobilized agonistic effector molecules may represent a new therapeutic tool to limit neutrophil hyperactivation and its sequelae in patients with severe immune disorders.

Recovery of neutrophil apoptosis by ectoine: a new strategy against lung inflammation

The life span of neutrophilic granulocytes has a determining impact on the intensity and duration of neutrophil driven lung inflammation. Based on the compatible solute ectoine, we aimed to prevent anti-apoptotic reactions in neutrophils triggered by the inflammatory microenvironment in the lung. Neutrophils from chronic obstructive pulmonary disease patients and control individuals were exposed to inflammatory mediators and xenobiotics in the presence or absence of ectoine. The in vivo relevance of this approach was tested in xenobiotic-induced lung inflammation in rats. The reduction of apoptosis rates of ex vivo-exposed neutrophils from all study groups was significantly restored in the presence of ectoine. However, natural apoptosis rates not altered by inflammatory stimuli were not changed by ectoine. Mechanistic analyses demonstrated the preventive effect of ectoine on the induction of anti-apoptotic signalling. Neutrophilic lung inflammation induced by single or multiple expositions of animals to environmental particles was reduced after the therapeutic intervention with ectoine. Analyses of neutrophils from bronchoalveolar lavage indicate that the in vivo effect is due to the restoration of neutrophil apoptosis. Ectoine, a compound of the highly compliant group of compatible solutes, demonstrates a reproducible and robust effect on the resolution of lung inflammation.

Dermal Application of Nitric Oxide In Vivo: Kinetics, Biological Responses, and Therapeutic Potential in Humans

Many local hemodynamic and vascular disorders may be the result of impaired bioavailability of nitric oxide (NO). Previous findings point to a therapeutic potential of dermal NO application in the treatment of hemodynamic disorders, but no reliable data are available on the mechanisms, kinetics, or biological responses relating to cutaneous exposure to NO in humans in vivo. Here we show that, owing to its excellent diffusion capacity, cutaneously applied NO rapidly penetrates the epidermal barrier in significant amounts, strongly enriching skin tissue and blood plasma with its vasoactive derivates. In parallel, it significantly increased vasodilatation and blood flow and reduced thrombocyte aggregation capacity. Data presented here for the first time show that, in humans, dermal application of NO has strong potential for use in the therapy of local hemodynamic disorders arising from insufficient availability of NO or its bioactive derivates.

Nano-coating protects biofunctional materials

The demand to develop convergent technology platforms, such as bio-functionalized medical devices, is rapidly increasing. However, the loss of biological function of the effector molecules during sterilization represents a significant and general problem. Therefore, we have developed and characterized a nano-coating (NC) formulation capable of maintaining the functionality of proteins on biological-device combination products. As a proof of concept, the NC preserved the structural and functional integrity of an otherwise highly fragile antibody immobilized on polyurethane during deleterious sterilizing irradiation (≥ 25 kGy). The NC procedure enables straight-forward terminal sterilization of bio-functionalized materials while preserving optimal conditioning of the bioactive surface.

Signalling-Dependent Adverse Health Effects of Carbon Nanoparticles Are Prevented by the Compatible Solute Mannosylglycerate (Firoin) In Vitro and In Vivo

The inhalation of combustion-derived nanoparticles leads to adverse health effects in the airways. In this context the induction of membrane-coupled signalling is considered as causative for changes in tissue homeostasis and pro-inflammatory reactions. The identification of these molecular cell reactions allowed to seek for strategies which interfere with these adverse effects. In the current study, we investigated the structurally different compatible solutes mannosylglycerate (firoin) from thermophilic bacteria and ectoine from halophilic bacteria for their capability to reduce signalling pathways triggered by carbon nanoparticles in target cells in the lung. The pre-treatment of lung epithelial cells with both substances decreased the particle-specific activation of mitogen-activated protein kinases and also the endpoints proliferation and apoptosis. Firoin applied into the lungs of animals, like ectoine, led to a significant reduction of the neutrophilic lung inflammation induced by particle exposure. The pro-inflammatory effect of carbon nanoparticles on human neutrophil granulocytes ex vivo was significantly reduced by both substances via the reduction of the anti-apoptotic membrane-dependent signalling. The data of this study together with earlier studies demonstrate that two structurally non-related compatible solutes are able to prevent pathogenic reactions of the airways to carbon nanoparticles by interfering with signalling events. The findings highlight the preventive or therapeutic potential of compatible solutes for adverse health effects caused by particle exposure of the airways.