Amyloid-␤(A␤)peptidesplayakeyroleinthepathogenesisofAlzheimer'sdiseaseandexertvarioustoxiceffectsonneurons;however,relatively littleisknownabouttheirinfluenceonglialcells.Astrocytesplayapivotalroleinbrainhomeostasis,contributingtotheregulationoflocalenergy metabolism and oxidative stress defense, two aspects of importance for neuronal viability and function. In the present study, we explored the effects of A␤ peptides on glucose metabolism in cultured astrocytes. Following A␤ 25-35 exposure, we observed an increase in glucose uptake and its various metabolic fates, i.e., glycolysis (coupled to lactate release), tricarboxylic acid cycle, pentose phosphate pathway, and incorporation into glycogen. A␤ increased hydrogen peroxide production as well as glutathione release into the extracellular space without affecting intracellular glutathione content. A causal link between the effects of A␤ on glucose metabolism and its aggregation and internalization into astrocytes through binding to members of the class A scavenger receptor family could be demonstrated. Using astrocyte-neuron cocultures, we observed that the overall modifications of astrocyte metabolism induced by A␤ impair neuronal viability. The effects of the A␤ 25-35 fragment were reproduced by A␤ 1-42 but not by A␤ 1-40 . Finally, the phosphoinositide 3-kinase (PI3-kinase) pathway appears to be crucial in these events since both the changes in glucose utilization and the decrease in neuronal viability are prevented by LY294002, a PI3-kinase inhibitor. This set of observations indicates that A␤ aggregation and internalization into astrocytes profoundly alter their metabolic phenotype with deleterious consequences for neuronal viability.

Neutrophil extracellular traps (NETs) contribute to innate immunity as well as numerous diseases processes such as deep vein thrombosis, myocardial ischemia, and autoimmune disease. To date, most knowledge on NETs formation has been gathered via the qualitative microscopic examination of individual neutrophils in vitro, or aggregate structures in vivo. Here we describe a novel flow cytometry (FLOW)-based assay to identify and quantify NETs using antibodies against key NETs constituents, specifically DNA, modified histones and granular enzymes. This method is applicable to both murine and human samples for the assessment of induced NETs in vitro, or detection of NETosis in vivo in blood samples. This FLOW-based method was validated by comparison with the well-established microscopy assay using two genetic mouse models previously demonstrated to show defective NETosis. It was then used on healthy human neutrophils for detection of ex vivo induced NETs and on blood samples from patients with sepsis for direct assessment of in vivo NET-forming neutrophils. This new methodology allows rapid and robust assessment of several thousand cells per sample and is independent of potential observer-bias, the two main limitations of the microscopic quantification. Using this new technology facilitates the direct detection of in vivo circulating NETs in blood samples and purification of NETting neutrophils by fluorescence-activated cell sorting (FACS) for further analysis.

Astrocytes play an important role in nervous system homeostasis. In particular, they contribute to the regulation of local energy metabolism and to oxidative stress defence. In previous experiments, we showed that long-term treatment with interleukin 1alpha (IL-1alpha) or tumor necrosis factor-alpha (TNFalpha) alone increases glucose utilization in primary culture of mouse astrocytes. In our study, we report that a combination of IL-1beta and TNFalpha exerts a synergistic effect on glucose utilization and markedly modifies the metabolic phenotype of astrocytes. Thus, IL-1beta+TNFalpha treated astrocytes show a marked decrease in glycogen levels, a slight but not significant decrease in lactate release as well as a massive increase in both the pentose phosphate pathway and TCA cycle activities. Glutamate-stimulated glucose utilization and lactate release, a typical feature of astrocyte energy metabolism, are altered after pretreatment with IL-1beta+TNFalpha. As far as mechanisms for oxidative stress defence are concerned, we observed that treatment with IL-1beta+TNFalpha decreases cellular glutathione content and increases glutathione release into the extracellular space while stimulating superoxide anion and nitric oxide production as well as H(2)O(2) release. Interestingly, stimulation of glucose utilization by IL-1beta+TNFalpha is not affected by the antioxidant N-acetyl-L-cysteine, suggesting that cellular stress does not account for this effect. Finally, the effects of cytokines on glucose utilization appear to involve multiple signaling cascades including the phosphoinositide 3-kinase and mitogen-activated protein kinases. Taken together these results establish that a proinflammatory environment such as observed in several neuropathological conditions including Alzheimer's disease, markedly modifies the metabolic phenotype of astrocytes.

Neutrophil extracellular trap (NET) formation can generate short-term, functional anucleate cytoplasts and trigger loss of cell viability. We demonstrated that the necroptotic cell death effector mixed lineage kinase domain-like (MLKL) translocated from the cytoplasm to the plasma membrane and stimulated downstream NADPH oxidase-independent ROS production, loss of cytoplasmic granules, breakdown of the nuclear membrane, chromatin decondensation, histone hypercitrullination, and extrusion of bacteriostatic NETs. This process was coordinated by receptor-interacting protein kinase-1 (RIPK1), which activated the caspase-8-dependent apoptotic or RIPK3/MLKL-dependent necroptotic death of mouse and human neutrophils. Genetic deficiency of RIPK3 and MLKL prevented NET formation but did not prevent cell death, which was because of residual caspase-8-dependent activity. Peptidylarginine deiminase 4 (PAD4) was activated downstream of RIPK1/RIPK3/MLKL and was required for maximal histone hypercitrullination and NET extrusion. This work defines a distinct signaling network that activates PAD4-dependent NET release for the control of methicillin-resistant (MRSA) infection.