P100 Atypical Anti-Neutrophil Cytoplasmatic Antibodies Are Antibodies Against Neutrophil Extracellular Traps: A Novel Pathophysiological Mechanism In Ulcerative Colitis

Abstract

Abstract Background The number of mucosal neutrophils, as well as the presence of neutrophil extracellular traps (NETs), correlate with disease activity in Ulcerative colitis (UC), an inflammatory bowel disease. Most (~80%) UC patients present atypical Anti-Neutrophil Cytoplasmatic Antibodies (a-ANCAs) to a yet unknown antigen, which shows a different immunofluorescence staining pattern when compared to cytoplasmatic (c-ANCA) and perinuclear (p-ANCA) that target proteinase (PR3) and myeloperoxidase (MPO), respectively. Here, we analyzed whether a-ANCAs specifically bind to NETs. Methods Blood neutrophils were treated to form non-lytic NETs using LPS-treated platelets with and without DNAse and Trypsin. Patient biopsies and in vitro-formed non-lytic NETs were incubated with p-ANCA, c-ANCA and a-ANCA positive patient and ANCA negative control serum and processed for immunofluorescence microscopy. Macrophage-mediated clearance of serum-pretreated NETs was analyzed in real-time using Incucyte microscopy. Results Ethanol-fixed neutrophils displayed c-ANCA, p-ANCA patterns, and a-ANCA web-like staining. Only c- and p-ANCA react to DNAse-treated ethanol-fixed neutrophils (Fig 1). Serum containing a-ANCA’s bound to non-lytic NETs that disappeared upon treatment with DNAse and Trypsin whereas c- and p- ANCA binding did not disappear upon DNAse treatment (Fig 2) indicating that both DNA and proteins are needed for a-ANCAs to bind to non-lytic NETs. In inflamed colonic tissue of UC patients, a-ANCA co-stained with extracellular DNA and neutrophil elastase covering the intestinal epithelium (Fig 3). In vitro, NETs were efficiently cleared by macrophages, which was strongly inhibited after pre-incubating with a-ANCAs (Fig 4). Macrophages exposed to a-ANCA-incubated NETs expressed higher CXCL-8 and IFN-B levels than the NETs exposed to control serum (both p<0.05). Figure 1 Figure 2 Figure 3 Figure 4 Conclusion a-ANCAs specifically bind to de novo DNA-protein antigens in non-lytic NETs, which prevents efficient macrophage-mediated clearance and induces a pro-inflammatory (M1) phenotype. This inflammatory milieu may contribute to the pathophysiology of UC.