Background/Purpose: Autoantibody-antigen complex-driven formation of neutrophil extracellular traps (NETs) is a major contributing factor to the pathophysiology and clinical manifestations of many different autoimmune diseases including SLE and RA. Inhibition of NET formation improves outcomes in animal models of autoimmune diseases. It has been previously shown that a neutrophil-resident proteinase inhibitor, serpin B1 (sB1) can regulate NET formation mediated by several agonists. However, it is currently unknown if sB1 can block immune complex (IC)-mediated NET formation. This study was designed to determine if sB1 can inhibit IC-mediated NET formation and if so, explore the molecular basis of the inhibition.

Methods: Recombinant forms of wild-type and variant sB1 that lacked protease-inhibitory activity or specifically inhibited elastase-like (neutrophil elastase [hNE], proteinase 3 [hPR3]) or chymotrypsin-like (e.g. cathepsin G [hCatG]) neutrophil serine proteases (NSP’s) were tested for their ability to block NET formation driven by RNP-containing ICs. In brief, human neutrophils were incubated in poly-L-lysine-coated tissue culture plates for 60 minutes in the presence of select reagents at varying concentrations prior to addition of ICs, PMA or A23187. After 4 hours of incubation, NETs were detached with micrococcal nuclease and analyzed for DNA content by staining with sytox green and peroxidase activity using the synthetic substrate TMB. Relative surface levels of CD66b, CD11b, and CD63, as well as ROS production, on treated neutrophils were measured by flow cytometry. Data were analyzed by FlowJo (Tree Star, Inc.).

Results: sB1 variants lacking protease inhibitory activity were unable to block NET formation. In contrast, the sB1 protein that could specifically inhibit chymotrypsin-like proteinases blocked NET formation whereas the sB1 protein that could specifically inhibit elastase-like proteinases did not. Consistently, the specific neutrophil elastase inhibitor had limited effect on NET formation, whereas the specific small molecule hCatG inhibitor also blocked NET formation providing further evidence implicating hCatG in the process of IC-driven NET formation. An oxidation resistant sB1 variant (C344A) effectively inhibited IC-mediated NET formation and was the only agent tested that significantly increased the levels of all three adhesion/activation markers on the surface of activated neutrophils. In addition, it significantly boosted intracellular ROS production.

Conclusion: sB1 can inhibit IC-mediated NET formation and does so in a hNE- and hPR3- independent manner. Our evidence is the first to suggest that hCatG released during neutrophil activation is directly involved in triggering IC-mediated NET formation. Future studies are needed to determine the role of sB1 on other neutrophil effector functions, including phagocytosis, adhesion and trafficking.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/serpin-b1-modulation-of-immune-complex-mediated-neutrophil-activation-and-net-formation-requires-its-chymotrypsin-like-inhibitory-activity/