Abstract: PO1462
Complement-Activated Polymorphonuclear Neutrophils Contribute to C3G Pathogenesis
Session Information
- Glomerular Diseases: Immunology and Inflammation in IgANP, C3GP, TMA, and Nephrotic Diseases
November 04, 2021 | Location: On-Demand, Virtual Only
Abstract Time: 10:00 AM - 12:00 PM
Category: Glomerular Diseases
- 1202 Glomerular Diseases: Immunology and Inflammation
Authors
- Maqsood, Maria, The Hospital for Sick Children, Toronto, Ontario, Canada
- Fine, Noah A., University of Toronto, Toronto, Ontario, Canada
- Ortiz, Carolina, The Hospital for Sick Children, Toronto, Ontario, Canada
- Walker, Patrick D., Arkana Laboratories, Little Rock, Arkansas, United States
- Licht, Christoph, The Hospital for Sick Children, Toronto, Ontario, Canada
Background
C3G is caused by dysregulation of the complement alternative pathway, but there is a gap in the pathogenetic cascade from complement activation (intravascular space) to inflammation and complement deposition in the glomeruli (extravascular space). Recently, biopsies have shown the presence of (activated) polymorphonuclear neutrophils (PMNs) in C3G glomeruli indicating an underappreciated role for PMNs in C3G pathogenesis.
Methods
PMNs were investigated in a transwell chamber allowing for different environments. Conditions were chosen to resemble the intravascular space (top well; serum containing) versus extracellular space (bottom well; serum-free conditions). PMNs were stimulated in the top well via various agonists, including complement, and allowed to transmigrate to the bottom well where they were monitored for the formation of Neutrophil Extracellular Traps (NETs) via immunofluorescence and SYTOX assay. Circulating C3G patient PMNs were examined for priming via flow cytometry.
Results
Upon complement stimulation, PMNs showed evidence for priming in serum conditions (top well). PMNs travelled to the bottom well following chemoattractant fMLP where they then completed the process of NET formation (NETosis) in serum-free conditions (bottom well). Results were validated ex vivo using C3G patient PMNs and autologous serum (Figure). In addition, incubation of control PMNs in C3G patient serum revealed a correlation between serum-albumin levels and the degree of NET formation. C3G patient PMNs showed upregulation of CD11b compared to controls.
Conclusion
Assigning a pathogenetic role to PMNs in C3G identifies a new treatment and monitoring strategy with the potential for improved long-term outcomes and quality of life.
PMNs were seeded in the top well of a transwell system and allowed to transmigrate for 12 h. PMNs from both wells were stained for IF with DAPI (blue), Citrullinated histone 3 (CitH3; green) and Myeloperoxidase (MPO; red). Scale bar 20 um. 63x magnification.