Background/Purpose: Systemic lupus erythematosus (SLE) replies on complement activation to drive many of the pathophysiologic features of disease. We and others have noted that SLE patients have constitutive activation of the complement component C3 (1). Elevated C3 activation products such as C3(H₂O) suggest that the alternative pathway is preferentially used to drive this process. Importantly, the production of C3(H₂O) is also critical for the complosome, the intracellular C3 activation pathway that confers functional changes in numerous cells types (2). Furthermore, prior work has shown that stimulated neutrophils can drive C3 activation, which in turn further activates neutrophils (3). As NETs are a key feature of SLE pathophysiology, we hypothesized that neutrophil extracellular traps (NETs) promote alternative pathway activation, producing C3(H₂O) which is in turn used to drive intracellular C3 activation.

Methods: To independently assess whether NETs can activate C3, we leveraged a recently developed acellular mimic of NETs, DNA-histone mesostructures (DHMs), that possess the ultrastructural and functional properties of NETs (4). Purified human native C3 and C2-depleted human serum (Complement Technologies, Tyler, Tx, USA) were added to DHMs or DNA-histone-free DHMs (control). C3(H₂O), factor Bb, C3a, C5a, and C5b-C9 were assessed using ELISA. Isolated human neutrophils were obtained from consented healthy controls and activated by either DHM-induced C3(H₂O) or phorbol myristate acetate (PMA), some in the presence of DNAse to digest NETs. Cell lysates were examined by western blot for intracellular C3 activation products (goat anti-human C3 polyclonal antibody, Complement Technologies). Images of neutrophils were obtained using a Zeiss LSM 880 Confocal with AiryScan.

Results: DHMs drove the rapid (< 5 minutes) and nearly complete ( >90%) activation of native C3 into C3(H₂O), suggesting a contact mechanism promotes early C3 activation. When C2-depleted was added to DHMs, products of alternative (Bb) and common pathway (C3a, C3a, C5b-9) were observed. When DHM-induced C3(H₂O) was added to primary neutrophils, it was readily taken up both unstimulated and PHA-activated neutrophils as observed by AiryScan confocal microscopy. Interestingly, only PHA-activated neutrophils were able to generate intracellular C3a and C3b products. Finally, using DNase-treated PHA-activated neutrophils to digest NETs, only the addition of DHM-induced C3(H₂O), and not native C3, induced C3 uptake, further supporting the role of NETs in C3 activation.

Conclusion: We found that NETs are sufficient to activate the alternative pathway of complement. The C3(H₂O) generated through this process was internalized by neutrophils, but only activated neutrophils were capable of intracellular C3 activation. Given the high NET load in SLE, we suspect that this may be the etiology for constitutive C3 activation observed in SLE patients.

References:

1. Kim et al, Arthritis Rheumatol, 2019, doi: 10.1002/art.40747
2. Elvington et al, J Clin Invest, 2017, doi: 10.1172/JCI89412
3. Camous et al, Blood, 2011, doi: 10.1182/blood-2010-05-283564
4. Weerappuli et al, Adv Healthc Mater, 2019, doi: 10.1002/adhm.201900926

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/neutrophil-extracellular-traps-are-sufficient-to-activate-the-alternative-pathway-of-complement/