230. NETs and terminal pathway factors as potential biomarkers for complement overactivation assessment in ANCA-associated vasculitis

Background: Clinical, in vitro, and animal model-derived evidence has demonstrated a critical involvement of the alternative complement pathway (aCP) in the pathogenesis of ANCA-associated vasculitis (AAV). In this regard, neutrophil extracellular traps (NETs) have been suggested to be a key element between ANCA-induced neutrophil activation and aCP. However, the role of the terminal complement pathway (tCP) is less well studied. Methods: A prospective, observational, multicenter study analyzing first episodes and relapses of patients with AAV, with a minimum follow-up of 6 months, was performed. Blood samples were collected at diagnosis (AAV-t1) and at remission (AAV-t2). Control population consisted of age and sex-matched individuals. Complement activation was assessed by analyzing the complement membrane attack complex (C5b-9) deposition on cultured endothelial cells (HMEC-1), by immunofluorescence, after exposing them to activated plasma (a-plasma: obtained by mixing patient’s citrated plasma with healthy subjects’ sera pool, 1:1). C5b-9 deposits induced by patients’ a-plasma were calculated as percentage of labeled area with respect to the total area analyzed. Results from patient and control samples were expressed as fold increase (mean±SEM) vs. those obtained with the pool of a-plasma from healthy subjects. Plasma levels of tCP and aCP soluble factors, such as sC5b-9 and sFBb (respectively), were also measured (mean±SEM). Circulating NETs were indirectly measured by quantifying circulant dsDNA plasmatic concentration (mean±SEM) as a NET surrogate. Results: The present results were obtained with samples from 13 AAV-MPO patients who achieved complete remission (38% men, age 63±14 years) and 10 controls (45% men, age 66±6 years). At AAV-t1, there was a statistically significant increase (p<0.05) of C5b-9 deposition on HMEC-1 in response to patients’ a-plasma (fold increase of 5.3±1.3) compared to control samples (fold increase of 1.2±0.2). Samples obtained at AAV-t2 induced less C5b-9 deposition than at AAV-t1 (fold increase of 0.9±0.2; p<0.05), with values similar to controls. Regarding soluble factors, levels of both sC5b-9 and sFBb were significantly increased in AAV-t1 (1882±418 ng/mL and 3.2±0.4 µg/mL, respectively; p<0.05) vs. AAV-t2 (852±104 ng/mL and 1.9±0.2 µg/mL, respectively; p<0.05). At AAV-t2, levels were similar to controls (708±42 ng/mL for sC5b-9, and 2.4±0.2 µg/mL for sFBb). Circulating NETs were also increased in AAV-t1 (22.2±3.5 µg/mL) compared to both AAV-t2 and controls (13.6±1.1 µg/mL and 13.7±0.3 µg/mL, respectively; p<0.05). Moreover, NETs in AAV-t1 presented a significant correlation with sFBb levels, both in AAV-t1 (r=0,709; p<0.05) and in AAV-t2 (r=0,585; p