The mechanism by which oxidative stress induces inflammation and vice versa is unclear but is of great importance, being apparently linked to many chronic inflammatory diseases. We show here that inflammatory stimuli induce release of oxidized peroxiredoxin-2 (PRDX2), a ubiquitous redox-active intracellular enzyme. Once released, the extracellular PRDX2 acts as a redoxdependent inflammatory mediator, triggering macrophages to produce and release TNF-α. The oxidative coupling of glutathione (GSH) to PRDX2 cysteine residues (i.e., protein glutathionylation) occurs before or during PRDX2 release, a process central to the regulation of immunity. We identified PRDX2 among the glutathionylated proteins released in vitro by LPS-stimulated macrophages using mass spectrometry proteomic methods. Consistent with being part of an inflammatory cascade, we find that PRDX2 then induces TNF-α release. Unlike classical inflammatory cytokines, PRDX2 release does not reflect LPS-mediated induction of mRNA or protein synthesis; instead, PRDX2 is constitutively present in macrophages, mainly in the reduced form, and is released in the oxidized form on LPS stimulation. Release of PRDX2 is also observed in human embryonic kidney cells treated with TNF-α. Importantly, the PRDX2 substrate thioredoxin (TRX) is also released along with PRDX2, enabling an oxidative cascade that can alter the -SH status of surface proteins and thereby facilitate activation via cytokine and Toll-like receptors. Thus, our findings suggest a model in which the release of PRDX2 and TRX from macrophages can modify the redox status of cell surface receptors and enable induction of inflammatory responses. This pathway warrants further exploration as a potential novel therapeutic target for chronic inflammatory diseases.cysteine oxidation | thiol oxidation | redox proteomics

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Protein cysteines can form transient disulfides with glutathione (GSH), resulting in the production of glutathionylated proteins, and this process is regarded as a mechanism by which the redox state of the cell can regulate protein function. Most studies on redox regulation of immunity have focused on intracellular proteins. In this study we have used redox proteomics to identify those proteins released in glutathionylated form by macrophages stimulated with lipopolysaccharide (LPS) after pre-loading the cells with biotinylated GSH. Of the several proteins identified in the redox secretome, we have selected a number for validation. Proteomic analysis indicated that LPS stimulated the release of peroxiredoxin (PRDX) 1, PRDX2, vimentin (VIM), profilin1 (PFN1) and thioredoxin 1 (TXN1). For PRDX1 and TXN1, we were able to confirm that the released protein is glutathionylated. PRDX1, PRDX2 and TXN1 were also released by the human pulmonary epithelial cell line, A549, infected with influenza virus. The release of the proteins identified was inhibited by the anti-inflammatory glucocorticoid, dexamethasone (DEX), which also inhibited tumor necrosis factor (TNF)-α release, and by thiol antioxidants (N-butanoyl GSH derivative, GSH-C4, and N-acetylcysteine (NAC), which did not affect TNF-α production. The proteins identified could be useful as biomarkers of oxidative stress associated with inflammation, and further studies will be required to investigate if the extracellular forms of these proteins has immunoregulatory functions.

Microbial infections are dependent on the panoply of interactions between pathogen and host and identifying the molecular basis of such interactions is necessary to understand and control infection. Phage display is a simple functional genomic methodology for screening and identifying protein-ligand interactions and is widely used in epitope mapping, antibody engineering and screening for receptor agonists or antagonists. Phage display is also used widely in various forms, including the use of fragment libraries of whole microbial genomes, to identify peptide-ligand and protein-ligand interactions that are of importance in infection. In particular, this technique has proved successful in identifying microbial adhesins that are vital for colonization.