Peripheral neutrophil phenotype and function in Ocular Mucous Membrane Pemphigoid.

Abstract

Purpose :
Neutrophils are recruited to infected sites and kill bacteria by phagocytosis, reactive oxygen species (ROS) production and the release of neutrophil extracellular traps (NETs). Neutrophils (Total Neutrophils (TN) and subsets (low density neutrophils (LDN), normal density neutrophils (NDN)) also have range of advanced functions including tumour initiation and growth, stimulation of proliferation, and tissue remodelling. We have previously shown that conjunctival neutrophils contribute towards progressive conjunctival scarring without visible inflammation seen in Ocular Mucous Membrane Pemphigoid (OcMMP). OcMMP is a heterogeneous group of autoimmune subepidermal blistering disorders that affect mucous membranes at the orifices where inflammation leads to progressive scarring. In this study, we examine the phenotype and function of peripheral neutrophils in OcMMP and hypothesize that these drive progressive blinding and conjunctival fibrosis.

Methods : Peripheral blood samples were obtained from patients with OcMMP (n=55) and healthy aged matched volunteers (n=16) at the Birmingham & Midland Eye Centre following informed consent. Phagocytic capacity, ROS production stimulated with Escherichia coli (E.coli) and NET formation stimulated with phorbol 12-myristate 13-acetate (PMA) and E.coli were analysed in TN, LDN and NDN using PhagoTest/Burst kits (Glycotope Biotechnology) and NETs assays. Ficoll hypaque segmentation was used to quantify the levels of LDN and NDN.

Results : A significantly reduced (p<0.05) phagocytic capacity and ROS production was observed after stimulating TN with E.coli. The results showed a significantly elevated levels of LDN (p<0.05) reduced phagocytic capacity and ROS production in LDN (p<0.05) and NDN of OcMMP patients. On stimulation with PMA, a high number of NETs was observed in TN, LDN and NDN cultures in comparison to E.coli stimulation (p<0.05).

Conclusions :
The results suggest that circulatory neutrophils, LDN and NDN in OcMMP patients have an altered function and a heterogeneous phenotype (activated, proinflammatory and immature) which may contribute towards conjunctival disease activity and progression.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.