Neutrophil granulocytes are important mediators of innate immunity, but also participate in the pathogenesis of (auto)inflammatory diseases. Neutrophils express a specific set of proteolytic enzymes, the neutrophil serine proteases (NSPs), which are stored in cytoplasmic granules and can be secreted into the extra- and pericellular space upon cellular activation. These NSPs, namely cathepsin G (CG), neutrophil elastase (NE), and proteinase 3 (PR3), have early been implicated in bacterial defense. However, NSPs also regulate the inflammatory response by specifically altering the function of cytokines and chemokines. For instance, PR3 and NE both inactivate the anti-inflammatory mediator progranulin, which may play a role in chronic inflammation. Here, we provide a concise update on NSPs as modulators of inflammation and discuss the biological and pathological significance of this novel function of NSPs. Mounting evidence support an important proinflammatory function for PR3, which may have been underestimated in the past.

Neutrophil extracellular traps (NETs) consist of antimicrobial molecules embedded in a web of extracellular DNA. Formation of NETs is considered to be a defense mechanism utilized by neutrophils to ensnare and kill invading pathogens, and has been recently termed NETosis. Neutrophils can be stimulated to undergo NETosis ex vivo, and are predicted to contain high levels of serine proteases, such as neutrophil elastase (NE), cathepsin G (CG) and proteinase 3 (PR3). Serine proteases are important effectors of neutrophil-mediated immunity, which function directly by degrading pathogenic virulent factors and indirectly via proteolytic activation or deactivation of cytokines, chemokines and receptors. In this study, we utilized a diverse and unbiased peptide library to detect and profile protease activity associated with NETs induced by phorbol-12-myristate-13-acetate (PMA). We obtained a "proteolytic signature" from NETs derived from healthy donor neutrophils and used proteomics to assist in the identification of the source of this proteolytic activity. In addition, we profiled each neutrophil serine protease and included the newly identified enzyme, neutrophil serine protease 4 (NSP4). Each enzyme had overlapping yet distinct endopeptidase activities and often cleaved at unique sites within the same peptide substrate. The dominant proteolytic activity in NETs was attributed to NE; however, cleavage sites corresponding to CG and PR3 activity were evident. When NE was immunodepleted, the remaining activity was attributed to CG and to a lesser extent PR3 and NSP4. Our results suggest that blocking NE activity would abrogate the major protease activity associated with NETs. In addition, the newly identified substrate specificity signatures will guide the design of more specific probes and inhibitors that target NET-associated proteases.