The conversion of native C3 to C3(H2O) is contact-driven facilitated by neutrophil extracellular traps

• Published in: Immunobiology (1979) Vol. 230; no. 4; p. 153049
• Main Authors: Sahu, Sanjaya Kumar, Weerappuli, Priyan D., Atkinson, John P., Kim, Alfred H.J.
• Format: Journal Article
• Language: English
• Published: Elsevier GmbH 01.07.2025
• ISSN: 0171-2985
• DOI: 10.1016/j.imbio.2025.153049

The tick-over of C3 is a slow, spontaneous, and rate-limiting process of the alternative pathway (AP) of complement. It maintains a low level (~1%) of baseline activity, ensuring the system is primed for rapid and efficient responses. It has been speculated that certain highly negatively charged surfaces such as polystyrene or air bubbles can drive the conversion of C3 to C3(H2O). Various autoimmune diseases are characterized by AP activation (i.e. lupus and antiphospholipid syndrome) along with high rates of NETosis. We speculate that elevated AP activation in these conditions may be due to high neutrophil extracellular trap (NET) load, creating excessive negatively charged surfaces that can drive C3(H2O) formation. To test this, we first examined whether NETs are sufficient to induce native C3 to C3(H2O) conversion. Bead-purified human neutrophils were treated with 100 nM phorbol 12-myristate 13-acetate (PMA) incubated for 2–3 hrs to induce NET formation, which was confirmed by immunofluorescence. DNA-histone mesostructures (DHMs) were also used, comprising of chromatin, histones, and trehalose, as “artificial” NETs with structural and biologic characteristics similar to isolated NETs (2). Native C3 was incubated with NETs or DHMs for 1, 2.5, or 5 minutes and C3(H2O) was quantified via ELISA (1). 1 unit/μL of deoxyribonuclease I (DNase I) was used to digest NETs over 15 minutes at 37 °C. We observed accelerated conversion (~1800-fold compared to an estimated 1% tickover rate)) of native C3 to C3(H2O) when C3 was co-incubated with NETs or DHMs, which was abrogated when DNase was added to NETs or trehalose only was used as controls. The rate of conversion was rapid, as the maximally observed C3(H2O) was reached at the earliest timepoint (1 min) and did not increase with additional incubation. Surprisingly, the rapid kinetics of conversion using DHMs was preserved when performed at +4 °C. These findings further support that the conversion of native C3 to C3(H2O) can be contact-mediated, and that biologic substrates such as NETs can dramatically facilitate this conversion. We are currently confirming the interaction between native C3 and NETs through cryo-EM structural determination. (1) Development and optimization of an ELISA to quantitate C3(H2O) as a marker of human disease. Front Immunol. 2019;10:703. PMID: 31019515 PMCID: PMC6458276. (2) Extracellular trap-mimicking DNA-histone mesostructures synergistically activate dendritic cells. Adv Healthc Mater. 2019;8(22): e1900926. PMID: 31614077 PMCID: PMC6872909.