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Abstract: FR-PO823

Elucidating the Effects of Complement Stress in Neutrophil Extracellular Traps (NETs) Formation in C3 Glomerulopathy

Session Information

Category: Glomerular Diseases

  • 1202 Glomerular Diseases: Immunology and Inflammation

Authors

  • Suntharalingham, Samuel Elijah, University of Toronto, Toronto, Ontario, Canada
  • Ortiz, Carolina, The Hospital for Sick Children, Toronto, Ontario, Canada
  • Feitz, Wouter J.c., The Hospital for Sick Children, Toronto, Ontario, Canada
  • Licht, Christoph, The Hospital for Sick Children, Toronto, Ontario, Canada

Group or Team Name

  • Licht Lab
Background

Neutrophil extracellular traps (NETs) have been recently implicated in several disease states, which is mostly attributed to their pro-inflammatory and prothrombotic properties. Neutrophils carry complement proteins, and when activated, release these complement proteins and contribute to their activation via NETs. By improving our understanding of the interaction of the complement system and neutrophils and their respective response to produce these NETs, we will be able to better understand how neutrophils and NETs play a role in complement-mediated diseases such as C3 glomerulopathy (C3G), in which both neutrophils and complement are expected to contribute to disease pathogenesis.

Methods

Neutrophils freshly isolated from healthy controls (HC) were used (male, ages 18-24 years old). Experiments were done at least threefold (N=3). We either applied our established protocol of complement activation via the use of a sensitizing antibody (monoclonal anti-human CD59 antibody) in combination with 50% NHS using healthy control (HC) neutrophils or investigated patient-derived neutrophils incubated in autologous serum or serum-free media. Complement deposition (C3b; C5b-9) on neutrophils was detected via IF and flow cytometry. NETosis was detected using SYTOXGreen assay and immunofluorescence (IF) imaging.

Results

We found that complement activation of neutrophils resulted in (i) surface deposition of C3b and C5b-9; (ii) appearance of citrullinated Histone 3 (cit-H3) and myeloperoxidase (MPO) release; (iii) the stepwise completion of NETosis after the transfer of neutrophils into serum-free media (SFM). In addition, we found that C3G patient-derived neutrophils (i) were positive for surface complement deposition; (ii) were positive for cit-H3; and (iii) completed NETosis after transfer from autologous serum into SFM. Finally, HC neutrophils (i) when incubated with C3G patient-derived serum showed cit-H3 formation, and (ii) NETosis when transferred into SFM.

Conclusion

Complement activation of neutrophils induces NETosis in a step-wise fashion with cit-H3 formation in serum (“priming”) and full NETosis in SFM. These findings can be interpreted as resemblance of an intra- vs. an extra-vascular environment, suggesting that neutrophils are primed intra-vascularly and committed to full NETosis only extra-vascularly.

Funding

  • Government Support - Non-U.S.