NEUTROPHIL EXTRACELLULAR TRAPS ARE SUFFICIENT TO ACTIVATE THE ALTERNATIVE PATHWAY OF COMPLEMENT, WHICH IS CONSTITUITIVELY ACTIVE IN HUMAN SLE

O023 / #182 Topic: AS14 - Innate Immunity ABSTRACT CONCURRENT SESSION 03: INNATE AND ADAPTIVE IMMUNITY IN SLE 22-05-2025 1:40 PM - 2:40 PM Background/Purpose Systemic lupus erythematosus (SLE) relies on complement activation to drive many of the pathophysiologic features of this disease. We noted that patients with SLE have constitutive activation of the complement component C3 via the alternative pathway.[1] The rate limiting step of alternative pathway activation is the conformational change of native C3 to C3(H 2 O), a process that is termed tickover and thought to be a spontaneous process. Additionally, prior work has shown that stimulated neutrophils can drive C3 activation, which in turn further activates neutrophils, producing neutrophil extracellular traps (NETs).[2] As NETs are a key feature of SLE pathophysiology, and activated neutrophils can activate C3, we hypothesized that NETs promote alternative pathway activation in SLE by promoting the conformational change of native C3 to C3(H 2 O). 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.[3] Purified human native C3 and C4-depleted human serum (Complement Technologies, Tyler, Tx, USA) were added to DHMs or DNA-histone-free DHMs (control). C3(H 2 O) and factor Bb were assessed using ELISA. Isolated human neutrophils were obtained from consented healthy controls and activated by phorbol myristate acetate (PMA), some in the presence of DNAse to digest NETs. Images of neutrophils were obtained using a Zeiss LSM 880 Confocal with AiryScan. Cryo-electron microscopy (cryo-EM) was applied to elucidate the atomic structures of native C3 and C3(H 2 O) using the Titan Krios G3 300kV Cryo-TEM. All processing of single-particle cryo-EM data was undertaken using cryoSPARK and RELION software. Results DHMs and isolated NETs from activated human neutrophils drove the rapid (< 5 minutes) and nearly complete (>90%) activation of native C3 into C3(H 2 O) with the formation of a product of alternative pathway activation Bb, suggesting a contact-mediated mechanism is sufficient to activate the alternative pathway (Figure 1). AiryScan confocal fluorescence microscopy confirmed the colocalization of native C3 with DHMs or NETs. Figure 1. The first atomic-level structural determination of C3(H 2 O) has been resolved to 3.20 angstrom (Figure 2A). Additionally, while native C3 that has been freeze-thawed once to create a mixture of native C3 and C3(H 2 O) revealed a structural intermediate that likely represents a metastable conformation termed C3(H 2 O)* that native C3 adopts as it converts to C3(H 2 O) (Figure 2B). Figure 2 Conclusions We found that NETs are sufficient to activate the alternative pathway of complement through the likely contact-mediated conversion of native C3 to C3(H 2 O). Given the high NET load and elevated serum iC3b levels (supporting alternative pathway activation) in SLE, we suspect that this may be the etiology for constitutive C3 activation observed in patients in SLE. We also generated the first atomic-level structural determination of C3(H 2 O) using cryo-EM, along with identification of a structural intermediate between native C3 to C3(H 2 O). These structures can be leveraged to identify small molecules that can interfere with alternative pathway activation, which may represent novel therapeutics in SLE and other alternative pathway-mediated diseases. References: [1.] Kim AHJ. Arthritis Rheumatol 2019;71:420-30. [2.] Camous L. Blood 2011;117:1340-9. [3.] Weerappuli PD. Adv Healthc Mater 2019;8:e1900926.

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