Neutrophil extracellular traps : function in infectious and non-Infectious conditions

Neutrophil extracellular traps (NETs) are composed of a backbone of chromatin, decorated with microbicidal peptides and proteolytic enzymes, and are used by neutrophils as a weapon against pathogens. Since their initial discovery in 2004, NETs have challenged scientists in terms of the nature of the triggers and the mechanisms of neutrophil release of NETs, as well as the implication of these extracellular structures in infectious and non-infectious human diseases. The work presented in this thesis, performed in an interdisciplinary perspective using an array of different methodologies, aims to deepen the knowledge on the functionality and dysfunctionality of NETs, including the mechanism of NET disposal and its immunological consequences. Once NETs have fulfilled their anti-microbial function, the timely clearance of these structures is needed in order to avoid a misguided autoinflammatory response. In Paper I, we showed that primary human macrophages employ TREX1, while dendritic cells use DNase1L3 to digest NETs purified from activated neutrophils. In addition, on the basis of cytokine profiling, we showed that NETs have immunomodulatory effects on phagocytes. In Paper II, we showed that JAGN1 is required for efficient fungal killing in NETs. Patients suffering from severe congenital neutropenia (SCN) present homozygous mutations in the gene encoding JAGN1 and are susceptible to bacterial and fungal infections. We found that JAGN1-deficient neutrophils isolated from an SCN patient, as well as neutrophil-like HL-60 cells with silenced JAGN1 expression, released NETs, but the expression of myeloperoxidase (MPO) was altered. In Paper III and IV we explored the interactions of NETs with graphene oxide (GO), a carbon-based 2-D material. Using ToF-SIMS, a mass spectrometry-based surface analytical method, we could show in Paper III that GO interacts with the plasma membrane of neutrophils, promoting cholesterol oxidation. In addition, we could define a size-dependent mechanism of GO induced release of NETs. Furthermore, we showed in Paper IV that GO undergoes efficient extracellular degradation through neutrophil degranulation or in NETs in a process dependent on MPO. Moreover, intermediate degradation products of GO did not cause DNA damage in lung cells. Overall, the work presented in this thesis has shed light on the clearance and degradation of NETs by phagocytic cells and the involvement of several endonucleases in a cell type-specific manner, and has confirmed the importance of NETs in fungal killing, with new evidence for a role of JAGN1 in this process. In addition, we have shown for the first time that GO triggers NETs and reported that GO may also undergo degradation in NETs in a similar manner as pathogens. Our studies have thus revealed that MPO expressed in NETs is a key element in eliminating infectious as well as non-infectious agents

Probing Substituents in the 1- and 3-Position: Tetrahydropyrazino-Annelated Water-Soluble Xanthine Derivatives as Multi-Target Drugs With Potent Adenosine Receptor Antagonistic Activity

Tetrahydropyrazino-annelated theophylline (1,3-dimethylxanthine) derivatives have previously been shown to display increased water-solubility as compared to the parent xanthines due to their basic character. In the present study, we modified this promising scaffold by replacing the 1,3-dimethyl residues by a variety of alkyl groups including combinations of different substituents in both positions. Substituted benzyl or phenethyl residues were attached to the N8 of the resulting 1,3-dialkyl-tetrahydropyrazino[2,1-f ]purinediones with the aim to obtain multi-target drugs that block human A1 and A2A adenosine receptors (ARs) and monoaminoxidase B (MAO-B). 1,3-Diethyl-substituted derivatives showed high affinity for A1 ARs, e.g., 15d (PSB-18339, 8-m-bromobenzyl-substituted) displayed a Ki value of 13.6 nM combined with high selectivity. 1-Ethyl-3-propargyl-substituted derivatives exhibited increased A2A AR affinity. The 8-phenethyl derivative 20h was selective for the A2A AR (Ki 149 nM), while the corresponding 8-benzyl-substituted compound 20e (PSB-1869) blocked A1 and A2A ARs with equal potency (Ki A1, 180 nM; A2A, 282 nM). The 1-ethyl-3-methyl-substituted derivative 16a (PSB-18405) bearing a m,p-dichlorobenzyl residue at N8 blocked all three targets, A1 ARs (Ki 396 nM), A2A ARs (Ki 1,620 nM), and MAO-B (IC50 106 nM) with high selectivity vs. the other subtypes (A2B and A3 ARs, MAO-A), and can thus be considered as a multi-target drug. Our findings were rationalized by molecular docking studies based on previously published X-ray structures of the protein targets. The new drugs have potential for the treatment of neurodegenerative diseases, in particular Parkinson's disease

Graphene Oxide Elicits Membrane Lipid Changes and Neutrophil Extracellular Trap Formation

Understanding the biological interactions of graphene-based materials is important for the safe use of these materials. Previous studies have explored the interaction between graphene oxide (GO) and macrophages but not the impact of GO on neutrophils, key cells of the immune system. Here, we synthesized GO sheets with differing lateral dimensions and showed by using an array of analytical and imaging techniques, including transmission and scanning electron microscopy, confocal microscopy, and time-of-flight secondary ion mass spectroscopy (ToF-SIMS), that GO elicited the formation of neutrophil extracellular traps (NETs). ToF-SIMS revealed pronounced perturbations of plasma membrane lipids, including a decrease in cholesterol and increased levels of oxidized cholesterol species. The induction of NETs was size dependent and associated with the production of mitochondrial reactive oxygen species and calcium influx. Importantly, antioxidant treatment reduced the production of NETs. These studies provide evidence that a previously undescribed biological effect of GO manifests through direct effects on membrane lipids. Graphene oxide (GO) is being investigated for various biomedical applications. Understanding the interactions between GO and living cells is of critical importance for the safe use of these materials in patients. In the present study, we identified effects of GO on neutrophils, the most common type of white blood cell. We first synthesized GO sheets of different sizes and carefully characterized the materials. Then, using various analytical and imaging techniques, we found that GO triggered so-called neutrophil extracellular traps or NETs. NETs are normally deployed by neutrophils to capture and destroy pathogens. We were able to show that GO caused significant changes in the lipid composition of the neutrophil cell membrane, whereby the oxidation of cholesterol set into motion a cascade of intracellular events leading to the formation of NETs. These studies show that GO acts directly on the neutrophil cell membrane and leads to the activation of a conserved anti-pathogen response. Graphene oxide (GO) is a promising material for a variety of biomedical and other applications. The increasing use of GO necessitates careful assessment of potential health hazards. Using primary neutrophils as a model, Mukherjee et al. show that GO elicits neutrophil extracellular traps. Furthermore, by using ToF-SIMS, the authors noted pronounced perturbations of plasma membrane lipids in cells exposed to GO

Graphene oxide is degraded by neutrophils and the degradation products are non-genotoxic

Graphene oxide (GO) undergoes neutrophil myeloperoxidase (MPO) dependent degradation and the degradation products are non-genotoxic for human lung cells.</p

Data_Sheet_1_Probing Substituents in the 1- and 3-Position: Tetrahydropyrazino-Annelated Water-Soluble Xanthine Derivatives as Multi-Target Drugs With Potent Adenosine Receptor Antagonistic Activity.PDF

<p>Tetrahydropyrazino-annelated theophylline (1,3-dimethylxanthine) derivatives have previously been shown to display increased water-solubility as compared to the parent xanthines due to their basic character. In the present study, we modified this promising scaffold by replacing the 1,3-dimethyl residues by a variety of alkyl groups including combinations of different substituents in both positions. Substituted benzyl or phenethyl residues were attached to the N8 of the resulting 1,3-dialkyl-tetrahydropyrazino[2,1-f ]purinediones with the aim to obtain multi-target drugs that block human A₁ and A_2A adenosine receptors (ARs) and monoaminoxidase B (MAO-B). 1,3-Diethyl-substituted derivatives showed high affinity for A₁ ARs, e.g., 15d (PSB-18339, 8-m-bromobenzyl-substituted) displayed a K_i value of 13.6 nM combined with high selectivity. 1-Ethyl-3-propargyl-substituted derivatives exhibited increased A_2A AR affinity. The 8-phenethyl derivative 20h was selective for the A_2A AR (K_i 149 nM), while the corresponding 8-benzyl-substituted compound 20e (PSB-1869) blocked A₁ and A_2A ARs with equal potency (K_i A₁, 180 nM; A_2A, 282 nM). The 1-ethyl-3-methyl-substituted derivative 16a (PSB-18405) bearing a m,p-dichlorobenzyl residue at N8 blocked all three targets, A₁ ARs (K_i 396 nM), A_2A ARs (K_i 1,620 nM), and MAO-B (IC₅₀ 106 nM) with high selectivity vs. the other subtypes (A_2B and A₃ ARs, MAO-A), and can thus be considered as a multi-target drug. Our findings were rationalized by molecular docking studies based on previously published X-ray structures of the protein targets. The new drugs have potential for the treatment of neurodegenerative diseases, in particular Parkinson's disease.</p

Thermodynamic analysis and optimization of an irreversible nano scale dual cycle operating with Maxwell-Boltzmann gas

In last decades, nano technology was developed. Since, nano scale thermal cycles will be possibly employed in near future. In this research, a nano scale irreversible dual cycle is investigated thermodynamically for optimization of performance. Ideal Maxwell-Boltzmann gas is used for working fluid in the system. It is chosen as working fluid. In this paper, two scenarios are introduced for optimization process. The outcomes of each of the scenarios are evaluated independently. Throughout first scenario, in order to maximize the dimensionless output work and first law efficiency of the system, multi-objective optimization algorithms are employed. Furthermore, in second scenario, two objective functions comprising the dimensionless output work are the dimensionless ecological function are maximized concurrently via employing multi objective optimization algorithms. The multi objective evolutionary approaches (MOEAs) on the basis of NSGA-II method are employed in this paper Decision making is done via three methods including LINAMP and TOPSIS and FUZZY. Finally, error analysis is implemented on the results obtained in this researc