97. Altered neutrophil phenotype and function in ANCA Vasculitis and COVID-19 highlights a disease specific response
Creators: name: Dwivedi1, Amrita
name: Mhaonaigh1, Aisling Ui
name: Batten1, Isabella
name: Finlay1, Conor
name: Phelan2, Stuart
name: Mark A Little1, 2
Publisher: name: Zenodo
Year: 2022

description: <strong>Background:</strong> Aberrant neutrophil activation is one of the key contributing factors in the ANCA vasculitis (AAV) immunopathology. A distinct subset of neutrophil is increased in AAV patients and associates with disease severity. This neutrophil subset is termed low-density granulocytes (LDGs), because they appear in the PBMC fraction of density separated blood, moreover, LDGs have been reported in COVID-19 (C-19). AAV and C-19 share similar pathways involved in disease mechanism such as high levels of neutrophil extracellular traps (NETs) and subsequent widespread endothelial dysfunction. We therefore hypothesize that these neutrophil subsets represent a disease specific response and possess distinct phenotypic and functional characteristics depending on the disease context. <strong>Methods</strong>: LDGs were isolated using a modified Percoll gradient preparation and analysed by multiparametric flow cytometry in patients with active and remission AAV, in mild and severe C-19 patients and in healthy controls (HC). The phenotyping panel included CD15, CD66b, CD16, CD10, CD62L, CD63, PD-L1, LOX-1, CXCR2 and CXCR4. We used UMAP dimensionality reduction technique to characterise the LDG populations within each cohort. We then assessed the production of reactive oxygen species (ROS) and NETs from LDGs following PMA and fMLP stimulation using dihydrorhodamine123 assay and DNA dye-based flow assay respectively. <strong>Results:</strong> We found increased levels of LDGs in AAV and C-19 patients which associated with disease severity. UMAP analysis of LDGs from remission AAV and mild C-19 yielded 1 major (over 70% of cells) and 3 minor (&gt;5%) sub-populations in each cohort. The phenotype of the predominant cluster identified was CD16<sup>+</sup>CD62L<sup>+</sup>CXCR2<sup>+</sup>CXCR4<sup>lo</sup>LOX-1<sup>-</sup>CD63<em><sup>+</sup></em> suggesting mature neutrophils. However, the LDG populations within acute AAV and severe C-19 were more complex. We identified 3 sub-populations in acute AAV: CD16<sup>+</sup>CD62L<sup>+</sup>CXCR2<sup>+</sup>CXCR4<sup>+</sup>LOX-1<sup>hi</sup>CD63<sup>+</sup>, CD16<sup>+</sup>CD62L<sup>-</sup>CXCR2<sup>+</sup>CXCR4<sup>-</sup>LOX-1<sup>hi</sup>CD63<sup>+ </sup>and CD16<sup>-</sup>CD62L<sup>-</sup>CXCR2<sup>-</sup>CXCR4<sup>+</sup>LOX-1<sup>hi</sup>CD63<sup>lo</sup>. Severe C-19 sub-populations comprised 2 major (&gt;20%, CD16<sup>-</sup>CD62L<sup>lo</sup>CXCR2<sup>lo </sup>and CD16<sup>hi</sup>CD10<sup>lo</sup>CD62L<sup>+</sup>LOX-1<sup>-</sup>) and 4 minor (&gt;10%) sub-populations. In addition to these phenotypic differences, we also observed different ROS and NET production capability in LDGs, especially enhanced NET production in fMLP stimulated LDGs from acute AAV. <strong>Conclusion:</strong> LDGs are significantly elevated in AAV and C-19, but their phenotypic and functional characteristics appear to be very different. Our results show that LDGs are comprised of several distinct sub-populations within acute AAV and severe C-19. AAV LDGs may possess enhanced functional response thereby suggesting a disease specific role for these neutrophil subsets. <strong>Disclosures:</strong> None