Background:The enzyme myeloperoxidase produces chlorine bleach at sites of inflammation. Results: 2-Thioxanthines are potent mechanism-based inactivators of myeloperoxidase. Conclusion: 2-Thioxanthines block production of chlorine bleach during inflammation. Significance: Mechanism-based inactivators of myeloperoxidase should limit oxidative stress at sites of inflammation.

Introduction Neutrophil accumulation is a well-established feature of Alzheimer’s disease (AD) and has been linked to cognitive impairment by modulating disease-relevant neuroinflammatory and vascular pathways. Neutrophils express high levels of the oxidant-generating enzyme myeloperoxidase (MPO), however there has been controversy regarding the cellular source and localisation of MPO in the AD brain. Materials and methods We used immunostaining and immunoassays to quantify the accumulation of neutrophils in human AD tissue microarrays and in the brains of APP/PS1 mice. We also used multiplexed immunolabelling to define the presence of NETs in AD. Results There was an increase in neutrophils in AD brains as well as in the murine APP/PS1 model of AD. Indeed, MPO expression was almost exclusively confined to S100A8-positive neutrophils in both human AD and murine APP/PS1 brains. The vascular localisation of neutrophils in both human AD and mouse models of AD was striking and driven by enhanced neutrophil adhesion to small vessels. We also observed rare infiltrating neutrophils and deposits of MPO around plaques. Citrullinated histone H3, a marker of neutrophil extracellular traps (NETs), was also detected in human AD cases at these sites, indicating the presence of extracellular MPO in the vasculature. Finally, there was a reduction in the endothelial glycocalyx in AD that may be responsible for non-productive neutrophil adhesion to the vasculature. Conclusion Our report indicates that vascular changes may drive neutrophil adhesion and NETosis, and that neutrophil-derived MPO may lead to vascular oxidative stress and be a relevant therapeutic target in AD.

The primary function of neutrophils is to engulf and destroy invading pathogens. If the bactericidal capacity of neutrophils is defective, an individual may suffer from enhanced susceptibility to potentially fatal microbial infection. To identify such defects, and to investigate the mechanisms used to kill bacteria, the bactericidal activity of neutrophils must be accurately quantified. This chapter provides details of a comprehensive microbiological technique that quantifies neutrophil bactericidal activity by measuring the loss of viability of ingested bacteria over time. Two variations of this technique are presented: a simple "one-step" protocol providing a composite measure of phagocytosis and killing, and a more advanced "two-step" protocol that allows calculation of separate rate constants for both of these processes.

MIF (macrophage migration inhibitory factor) plays a central role in the promotion and maintenance of the inflammatory response. It is implicated in a number of inflammatory diseases including sepsis, arthritis and colitis, and in diseases with an inflammatory component, such as atherosclerosis, diabetes and cancer. MIF has an unusual N-terminal proline with catalytic activity, and targeting of this residue by small-molecule inhibitors has been shown to interfere with the biological activity of MIF. The objective of the present study was to determine if MIF was susceptible to modification by epicatechins, a group of dietary flavonoids with known anti-inflammatory properties. Epicatechins are substrates for peroxidases including neutrophil-derived MPO (myeloperoxidase). In the present study we show that oxidation of the catechol moiety of epicatechins to an ο-quinone by MPO generates potent MIF inhibitors. Near complete inhibition of MIF by the MPO/H2O2/epicatechin system was achieved at equimolar concentrations of epicatechin and MIF, even in the presence of other MPO substrates. We have characterized the modification introduced by oxidized (-)-epicatechin on MIF by LC-MS (liquid chromatography MS) and found it to occur at the N-terminal proline. We propose that MIF inhibition by oxidized epicatechins contributes to the anti-inflammatory activity of these compounds.

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. ABSTRACTCalprotectin provides nutritional immunity by sequestering manganese and zinc ions. It is abundant in the lungs of patients with cystic fibrosis but fails to prevent their recurrent infections. Calprotectin is a major protein of neutrophils and composed of two monomers, S100A8 and S100A9. We show that the ability of calprotectin to limit growth of Staphylococcus aureus and Pseudomonas aeruginosa is exquisitely sensitive to oxidation by hypochlorous acid. The N-terminal cysteine residue on S100A9 was highly susceptible to oxidation which resulted in cross-linking of the protein monomers. The N-terminal methionine of S100A8 was also readily oxidized by hypochlorous acid, forming both methionine sulfoxide and the unique product dehydromethionine. Isolated human neutrophils formed these modifications on calprotectin when their myeloperoxidase generated hypochlorous acid. Up to 90% of the N-terminal amine on S100A8 in bronchoalveolar lavage fluid from young children with cystic fibrosis was oxidized. Oxidized calprotectin was higher in children with cystic fibrosis compared to disease controls, and further elevated in those patients with infections. Our data suggest that oxidative stress associated with inflammation in cystic fibrosis will stop metal sequestration by calprotectin. Consequently, strategies aimed at blocking extracellular myeloperoxidase activity should enable calprotectin to provide nutritional immunity within the airways.

Background: Neutrophils generate oxidants to kill microbes ingested into phagosomes, but extraphagosomal protein oxidation may also occur. Results: Specific neutrophil cytosolic proteins were carbonylated following phagocytosis; carbonylation was impaired by myeloperoxidase inhibitors or radical scavengers that inhibit lipid peroxidation. Conclusion: Lipid peroxidation in the phagosomal membrane leads to oxidation of cytosolic proteins. Significance: Protein oxidation may influence the function and fate of post-phagocytic neutrophils.

This chapter describes two methods for measuring the bactericidal activity of neutrophils. These are a new simple fluorescence-based assay, which quantifies bactericidal activity by measuring changes in bacterial fluorescence associated with a loss of membrane potential over time, and a more traditional colony counting protocol. Two variations of these techniques are presented: a "one-step" protocol providing a composite measure of phagocytosis and killing, and a "two-step" protocol that allows calculation of separate rate constants for both of these processes.

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