122. Molecular Imaging to Identify Neutrophil Activation in ANCA-Vasulitis
Creators: name: Schmider1, Angela
name: Giorgianna Ellis1
name: Soberman1, Roy
name: Zonozi2, Reza
name: Niles2, John
Publisher: name: Zenodo
Year: 2022
Subjects: Vasculitis
ANCA
Abstracts
Giant Cell Arthritis
MPA
GPA
EGPA
Takayasu
IgA vasculitis

description: <strong>Background:</strong> Anti-neutrophil cytoplasmic antibody (ANCA)-vasculitis (AAV) is a small-vessel vasculitis caused by pathogenic autoantibodies directed against proteinase 3 (PR3) or myeloperoxidase (MPO) (1). Leukotrienes (LT)s are bioactive signaling lipids derived from arachidonic acid (AA) (2, 3) and have been suggested to play a role in inflammatory diseases (4), acting as a signal relay molecule that increases recruitment range and enhances directional migration (5). LTs initiate and amplify innate and adaptive immune responses made by leukocytes and play a central role in their recruitment to inflamed sites (6,7). We have shown that 5-lipoxygenase (5-LO), the 5-lipoxygenase-activating protein (FLAP), and LTC<sub>4</sub> synthase are assembled in novel macromolecular complexes on the nuclear envelope to initiate LT synthesis (8-12). 5-LO is a cytosolic protein that moves to the nuclear envelope in a calcium dependent manner. FLAP is an integral membrane protein that behaves as a scaffold protein to present AA to 5-LO. Mast cells and monocytes generate LTC<sub>4</sub> and LTB<sub>4</sub> while neutrophils only synthesize LTB<sub>4</sub>. Chemoattractants recruit neutrophils out of the peripheral circulation to inflamed tissues and interactions between complement, neutrophils and ANCA contribute to the onset and amplification of AAV (13,14). C5a and its receptor, C5aR, play a central role in ANCA-mediated neutrophil recruitment and activation. We showed that priming healthy human neutrophils with granulocyte macrophage-colony stimulating factor (GM-CSF) prior to C5a stimulation produces LTB<sub>4</sub> in a time-dependent manner. Figure 1 illustrates the evolution of the following neutrophil states: 1) resting state, 2) following C5a-priming and antigen translocation to the surface, ANCA and LTB<sub>4</sub> bind 3) activated state and transmigration­. Our goal is to leverage a new understanding of lipids and protein-protein interactions using novel molecular imaging techniques to predict disease activity in AAV. <strong>Methods</strong>: <em>Cell Isolation and treatment:</em> Neutrophils were isolated from peripheral blood of healthy donors and from patients with active AAV. Cells were left untreated or primed with 1 ng/mL GM-CSF for 45 min then treated with 50 nM C5a for 0, 2, 4, 6, 8 and 10 min. <em>Imaging and analysis:</em> Following treatment, cells were prepared for direct Stochastic Optical Reconstruction Microscopy (dSTORM) combined with computational analysis (Clus-DoC), Fluorescence Lifetime Imaging Microscopy (FLIM) and Enzyme Immunosorbent Assays (EIA). <strong>Results:</strong> EIA data show that LTB<sub>4</sub> production was minimal in cells untreated from a healthy donor (45 pg/ml), however after priming and activation levels increased to 280 pg/ml. Comparatively, LTB<sub>4</sub> levels in untreated neutrophils from a patient with active disease started at 1484 pg/ml and increased to 1980 pg/ml following priming and activation, suggesting that peripheral neutrophils in patients with active AAV are pre-activated and are making LTB<sub>4</sub>. STORM illustrates 5-LO and FLAP association on the nuclear envelope of neutrophils from a representative healthy donor only after priming and cell activation. Neutrophils from a representative patient with AAV show 5-LO and FLAP associated in both untreated and primed and activated conditions. FLIM revealed that in neutrophils from healthy donors that were primed with GM-CSF and activated with C5a for 0, 2, 4, 6, 8 and 10 min, the FLIM t<sub>1</sub> values decreased (suggesting a close interaction) at the same time LTB<sub>4</sub> levels increased. In contrast, neutrophils from patients with AAV that were primed with GM-CSF and activated with C5a for 0, 2, 4, 6, 8 and 10 min, the FLIM t<sub>1</sub> values remained constant (suggesting no change) and priming and cell activation did not synthesize LTB<sub>4</sub>. <strong>Conclusions</strong>: Circulating neutrophils from patients with AAV are pre-activated defined by the interaction of 5-LO:FLAP on the nuclear membrane and subsequent production of LTB<sub>4</sub>. <strong>Disclosures: </strong>None. <strong>Figure 1. </strong>Model of the LTB<sub>4</sub>/C5a amplification loop in neutrophil transmigration (created by Schmider).