Does NLRP3 Inflammasome assembly in neutrophils support ferritin-mediated inflammation and NETosis?

• Applicant: Dr. Viktoria Elisabeth Maria Schindler
• Subject Area: Rheumatology ; Clinical Immunology and Allergology
• Term: since 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 529990128

Adult Onset Stills Disease (AOSD) is an autoinflammatory syndrome characterized by severe systemic inflammation with a variable clinical presentation. Inflammation in AOSD is marked by extremely elevated serum ferritin levels. It is therefore considered part of the hyperferritinemic syndromes. The excessive inflammatory response in hyperferritinemic diseases leads to a massive release of proinflammatory cytokines resulting in cytokine storm. Alongside hyperferritinemia, peripheral neutrophilia and elevation of the inflammasome cytokines IL-1 and IL-18 are observed in AOSD. NLRP3 is a pattern recognition receptor that recognizes pathogens and endogenous danger signals intracellularly, resulting in the formation of the NLRP3 inflammasome. The NLRP3 inflammasome, especially in macrophages, is believed to play an important role in the inflammatory processes in AOSD. In recent years it has become increasingly clear that NLRP3 inflammasome signaling also takes place in neutrophils. NLRP3 is involved in a form of organized cell death in neutrophils called NETosis leading to the release of neutrophil extracellular traps (NETs), which are networks of decondensed chromatin. NETosis can be enhanced by various triggers including ferritin. The extent of neutrophil involvement in the development of cytokine storm and the importance of NLRP3 hereby are not yet known. The proposed project aims to elucidate the role of NLRP3 in ferritin-mediated neutrophil inflammation. We seek to determine whether ferritin-triggered NETosis and NLRP3 inflammasome activation in neutrophils are interdependent and result in the secretion of IL-1. The central hypothesis of this study is that ferritin-mediated inflammation is NLRP3-dependent and that the neutrophil is the essential player in this process. To investigate this a mouse model of ferritin-induced inflammation will be used. The inflammatory response will be characterized by cell counts, cytokine measurement, and morphological features such as hepatosplenomegaly. The significance of neutrophils will be demonstrated by the depletion and activation of neutrophils. The influence of ferritin on inflammasome activation and NET formation will be studied in isolated neutrophils. NETosis will be visualized by immunofluorescence and measurement of cell-free DNA. Immunofluorescence and levels of caspase-1 and IL-1 production will be used as indicators of inflammasome activation. To determine the importance of NLRP3, the inflammatory effects of ferritin will be examined in NLRP3 wild-type and NLRP-deficient mice and confirmed in vitro. The role of NLRP3 will be substantiated by the pharmacological inhibition of NLRP3. The proposed project is expected to shed new light on the underlying mechanisms of ferritin-mediated inflammation and to provide a scientific foundation for future projects. Evaluating the role of NLRP3 in AOSD could provide a new target for novel therapeutic agents.

• DFG Programme: WBP Fellowship
• International Connection: USA
• Host: Professorin Denisa D. Wagner, Ph.D.