Redox Regulation of Neutrophil Extracellular Traps Formation and IL-1β Processing in Serum-induced Arthritis

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Student thesis: Master's Thesis

Abstract

Neutrophil extracellular traps (NETs) are structures of chromatin filaments decorated with histones proteases and granular proteins The release of NETs also known as NETosis expose DAMP signals autoantigens proteases and reactive oxygen species (ROS) to the extracellular milieu and often lead to tissue damage and inflammation However it is also reported that serine protease released along with NETs are capable of degrading pro-inflammatory mediators and subsequently resolve inflammation In our previous studies we discovered that ROS regulate serum-induced arthritis a murine model for immune-mediated arthritis including rheumatoid arthritis (RA) ROS regulate arthritic inflammation via suppressing cathepsin B which mediates IL-1β maturation in the joints Moreover the production of ROS is critical for NET formation As a result we postulated that the development of severe joint inflammation in ROS-deficient mice is due to insufficient generation of ROS and NETs which revoke the degradation of pro-inflammatory cytokines by NET-derived proteases Hence we set out to investigate ROS regulations in NET-mediated cytokine processing in autoimmune arthritis In this study we treated wild-type mice and ROS-deficient mice Ncf1-/- with K/BxN arthritogenic mice serum Disease severity was monitored with IVIS Spectrum micro-CT and physical measurements In order to investigate whether K/BxN serum can induce NETosis neutrophils were incubated with K/BxN serum and phorbol-12-myristate-13-acetate (PMA) to induce NETs formation In addition to identify NETosis we also conducted experiments to establish a feasible approach to isolate NETs while preserving the protease activity and biological function Our results showed that even though ROS signals were only seen in wild-type mice joint swelling was significantly more severe in Ncf1-/- mice compared with wild-type controls Moreover significant bone erosion and bone mineral density loss were found in Ncf1-/- mice when compared with those in wild-type mice These data support the hypothesis that ROS is anti-inflammatory in serum-induced arthritis We also showed with fluorescence microscopy that large amount of extracellular DNA stretches were generated after K/BxN serum and PMA were added In the final part we demonstrated that NET fragments can be isolated from the neutrophil-NET meshes while retain its protease activity and biological function Furthermore NET-derived neutrophil elastase was active in processing pro-IL-1β to the mature form In conclusion our data gives awareness of the association between NETs ROS and RA Lacking ROS may lead to insufficient NET formation and cause deteriorating inflammation Even though the methodology of NET isolation have been proved to be tricky we successfully establish an approach which yields sufficient amount of viable NET sample which will be applied to the future experiments regarding the mechanism of rheumatoid arthritis and may aid the design of effective strategies to control NET-associated diseases
Date of Award2018 Jul 18
Original languageEnglish
SupervisorChi-Chang Shieh (Supervisor)

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