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Abstract: FR-PO1074

Elucidating the Role of Neutrophil Extracellular Traps in Atypical Hemolytic Uremic Syndrome

Session Information

Category: Glomerular Diseases

  • 1202 Glomerular Diseases: Immunology and Inflammation

Authors

  • Suntharalingham, Samuel Elijah, University of Toronto, Toronto, Ontario, Canada
  • Ortiz, Carolina, Hospital for Sick Children, Toronto, Ontario, Canada
  • Licht, Christoph, The Hospital for Sick Children, Toronto, Ontario, Canada
Background

About 10% of patients manifest hemolytic uremic syndrome (HUS) without toxins or diarrhea. These cases are referred to as atypical HUS (aHUS) and occur due to a dysregulated complement system leading to unchecked complement activation on the vascular endothelium. Genetic mutations account for only 50-60% of aHUS cases only with a large variation in the age and severity of the disease phenotype. For the past decade, neutrophils have been shown to produce neutrophil extracellular traps (NETs), which are associated with proinflammatory and prothrombotic properties. We propose that NETs are involved in aHUS pathogenesis.

Methods

Resting neutrophils freshly isolated from healthy donors were used (male, ages 18-24 years old). Experiments were done at least threefold (N=3). We have previously shown that neutrophils contain complement components, which they deposit on NETs, and that NETs contribute to complement activation. In this study, we aim to determine if NETosis can be induced by complement activation. In order to activate complement, neutrophils were sensitized via a monoclonal anti-human CD59 (x30 min) followed by incubation with 50% human serum as source of complement (x4 h). Immunofluorescence (IF) imaging was performed to visualize NETosis and complement activation. A SYTOX Green assay was used to quantify the kinetics of NETs formation.

Results

We found that: 1) the use of our complement sensitization protocol resulted in C3b deposition and C5b-9 complex formation on neutrophils; 2) NETosis could be induced in a complement-dependent manner; 3) both citrullinated Histone 3 (cit-H3) and myeloperoxidase (MPO) are both present in the NETs of complement-stimulated neutrophils.

Conclusion

Complement activation taking place on the surface of neutrophils induces NETosis, a new finding pointing towards a crucial role for neutrophils in the pathogenesis of complement-mediated TMA.

Funding

  • Government Support - Non-U.S.