Defining the phenotype and distribution of resident immune cells surrounding meibomian gland orifices in mice

Abstract

Purpose : Inflammation and keratinisation of the meibomian glands (MGs) and MG orifices (MGOs) are pathophysiological mechanisms involved in MG dysfunction (MGD). However, the normal immune cell (IC) profile of MGOs, which act as an interface between the external environment and internal glandular duct, has not been clearly defined. The aim of this study was to determine the presence, phenotype and distribution of MG orifice-associated immune cells (MOICs) in healthy young and aged mice.

Methods : Flatmounts of the superior and inferior eyelids of young (i.e. 12-15-week old; n=9-12/group) and older (i.e. 21-29-week old; n=5-6/group) CD11c^eYFP and Cx3cr1-deficient Cx3Cr1^GFP/GFP mice were used. CD11c⁺ dendritic cells (DCs) and Cx3cr1⁺ ICs surrounding MGOs were visualized using four-colour immunofluorescent confocal microscopy. Eyelids were incubated with antibodies targeting MHC-II⁺ ICs, and F-actin to visualise MGOs. MOIC density (cells/mm²) within a normalized peri-orifice area, relative to the MGO area, were quantified. Two adjacent MGOs in the nasal, central and temporal regions of both superior and inferior eyelids were analysed.

Results : In descending order of average density, CD11c⁺ DCs (abundant), MHC-II⁺ and Cx3cr1⁺ (scarce) ICs were identified at MGOs. More MOICs expressed CD11c⁺ nasally than temporally in young mice (nasal 942±391 vs temporal 680±348 cells/mm², p=0.0199), but not in older mice (nasal 932±592 vs temporal 611±446 cells/mm² p>0.05). On average, there were more Cx3cr1⁺ IC nasally compared with temporally in older mice (nasal 93±43 cells/mm² vs temporal 5±0.2 cells/mm², p=0.048). In young mice, there were no regional differences in MHC-II⁺ IC density along the eyelid, but fewer MOICs expressed MHC-II⁺ in young Cx3cr1-deficient mice (186±248 cells/mm²) compared to CD11c^eYFP mice (350±280 cells/mm²; p=0.0048).

Conclusions : Immune cells that surround mice MGOs are mostly CD11c⁺ and MHC-II⁺, with few expressing Cx3cr1⁺. There are more CD11c⁺ DCs in MGOs at the nasal eyelid, relative to the temporal region, in young mice. The Cx3cr1 receptor may have a partial role in the presence of MHC-II⁺ ICs around MGOs, as fewer MOICs expressed MHC-II⁺ in young Cx3Cr1^GFP/GFP mice. These novel findings provide a basis for further investigations of MOICs and their role in MGD.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.