POS0252 ANCA IGG PROMOTION OF NEUTROPHIL RECRUITMENT, MIGRATION AND VASCULAR DAMAGE IN ANCA ASSOCIATED VASCULITIS MAY BE ENHANCED BY HYPOXIA
Authors: N. Pisacano, A. Dhutia, S. Rothery, S. P. Mcadoo, C. D. Pusey, E. Chilvers, A. Cowburn, K. Lodge, M. Prendecki
Journal: Annals of the Rheumatic Diseases (2024)

<h3>Background:</h3> Neutrophils are the main mediators of disease in ANCA associated vasculitis (AAV). The effect of pathogenic ANCA IgG on neutrophils promotes neutrophil adhesion and migration at the endothelium, in addition to tissue infiltration. NETs are present in patients with active AAV (aAAV), and vascular inflammation and occlusion occurs as a result. Sites of inflammation and tissue injury are presumably hypoxic in AAV, and whether pathological hypoxia (1% O<sub>2</sub>) enhances these effects remains unknown. <h3>Objectives:</h3> Determine whether 1% O<sub>2</sub> alters the effect of ANCA on neutrophil protein secretion, transendothelial migration (TEM), and NET formation in comparison to normoxia (21% O<sub>2</sub>). <h3>Methods:</h3> Neutrophils were isolated from healthy controls (HC) and patients with aAAV using immunomagnetic separation. For TEM and protein secretion, glomerular endothelial cells (gEC) were cocultured with neutrophils from HC for 1.5h to assess neutrophil TEM towards chemoattractant (IL-8, 200 pg/mL) and protein secretion (without IL-8 treatment) at 21% O<sub>2</sub> or 1% O<sub>2</sub>. Isolated neutrophils were primed (TNF) and stimulated with MPO- or PR3-ANCA IgG or control IgG (CIgG) in 21% or 1% O<sub>2</sub>. A positive control of TNF primed gEC (10 ng/mL, 24h) were used. Transmigrated neutrophils were counted via haemocytometer. Protein secretion levels in supernatant measured by ELISA. NETosis was visualised by stimulating neutrophils in chamber slides and staining for H3Cit. For RNA-Sequencing (RNA-Seq), cells were lysed, RNA was extracted, and samples were sequenced using poly(A) RNA-Seq. <h3>Results:</h3> At 21% O<sub>2</sub>, TEM across a gEC monolayer towards chemoattractant (IL-8) increased following MPO- and PR3-ANCA stimulation compared to untreated neutrophils (Figure 1A; MPO: p=0.0017, PR3: p=0.0021). At 1% O<sub>2</sub>, there was a significant increase in neutrophil TEM in all conditions in comparison to 21% O<sub>2</sub>. Increased IL-8 secretion was seen in MPO- and PR3-ANCA stimulated coculture at 21% O<sub>2</sub> (MPO: p=0.0004, PR3: p=0.0003). This was enhanced by incubation at 1% O<sub>2</sub> (MPO: p=0.0003, PR3: p=0.0001) (Figure 1B). Similar results were seen for ICAM-1 secretion (Figure 1B). MPO- and PR3-ANCA stimulation increased NET formation at 21% O<sub>2</sub> in comparison to untreated and CIgG stimulated neutrophils and this effect was enhanced by incubation at 1% O<sub>2</sub> (Figure 1C). RNA-Seq identified IL-8 (<i>CXCL8</i>), <i>ICAM-1</i>, and Tissue Factor (<i>F3</i>) being significantly upregulated differentially expressed (DE) genes (p&lt;0.05, Log<sub>2</sub>FC &lt;0.05) in MPO- and PR3-ANCA stimulated neutrophils at 21% and 1% O<sub>2</sub> in comparison to untreated cells. <i>CD177</i> and <i>PRTN3</i> were significantly upregulated DE genes in isolated neutrophils from patients with aAAV in comparison to HC (data not shown). <h3>Conclusion:</h3> In comparison to untreated neutrophils, isolated HC neutrophils stimulated with ANCA undergo increased TEM across a gEC monolayer, increased protein secretion (IL-8 and ICAM-1) in neutrophil-gEC co-culture, increased NET formation, and upregulation of gene expression of <i>IL-8</i>, <i>ICAM-1</i>, and <i>F3</i>. This pathogenic effect of ANCA shown at 21% O<sub>2</sub><i>in vitro</i> is enhanced when neutrophils are incubated <i>in vitro</i> at 1% O<sub>2</sub>. Increased adhesion factors are present in the renal glomeruli, and increased neutrophil CD177 and PRTN3 expression in aAAV facilitates TEM through interaction with PECAM-1 (1). The effect of ANCA and hypoxia regulated increased TEM in HC neutrophils may therefore be more robust in aAAV. In aAAV, hypoxia may enhance ANCA promoted neutrophil induced endothelial injury. <h3>REFERENCES:</h3> [1] Sachs UJH, Andrei-Selmer CL, Maniar A, Weiss T, Paddock C, et al. The neutrophil specific antigen CD177 is a counter-receptor for platelet endothelial cell adhesion molecule-1 (CD31). J Biol Chem. 2007; 282(32): 23603-12. <h3>Acknowledgements:</h3> <b>NIL.</b> <h3>Disclosure of Interests:</h3> <b>None declared.</b>