Abstract
Purpose :
Conjunctival goblet cells (GCs) contribute to ocular immune homeostasis via Thrombospondin-1 (TSP-1)-mediated TGFβ2 activation, which in turn modulates dendritic cell (DC) phenotype by decreasing expression of activation markers, including MHC class II. This study determined the location of antigen presenting cells (APCs) in the conjunctiva and tested the hypothesis that microbial product flagellin (FL)-mediated GC responses disrupt homeostasis to promote APC activation that leads to corneal barrier damage.
Methods :
Conjunctival explants or frozen sections from WT (C57BL/6) and TSP-1 -/- mice were immunostained for CD11c, MUC5AC, MHC class II or TSP-1 and examined by confocal or fluorescence microscopy. WT mice were treated with FL (10 ng) topically for 7 days. Corneal barrier was monitored for 4 weeks with fluorescein staining. Primary cultured GCs were treated with FL (0-10mg/ml) or heat-killed pathogenic S. aeruginosa strain PA14 for 24 hr. Levels of IL-6 and active TGFβ were determined in culture supernatants using ELISA and bioassay respectively. Message for TSP-1 was determined by real-time PCR.
Results :
In the conjunctiva CD11c+ cells were located adjacent to MUC5AC+ GCs. Their cell bodies in the stromal layer, immediately below the epithelial layer, extended processes across the epithelium in TSP-1-/- conjunctiva previously reported to harbor increased microbial frequency. Such processes were not detected in WT tissue. Frequency of CD11c+MHC class II+ cells was increased in FL-treated conjunctiva compared to untreated controls. Primary GC cultures responded to PA14 and FL with an increased IL-6 secretion compared to controls (1193±58.5, 884±60.6 vs. 5.6±0.2 respectively, p<0.05). FL-stimulated GCs secreted reduced active TGFβ consistent with their reduced expression of TSP-1 message (1715±40.7 vs. 4459±75.2, p<0.05) and immunostaining in FL-treated conjunctiva. These changes correlated with an increased fluorescein staining score 4 weeks after FL treatment compared to the baseline (8.3±0.5 vs. 2.8±0.6, p<0.05).
Conclusions :
Our results demonstrate that DCs in the conjunctiva can extend trans-epithelial processes, presumably to sample microbes at the ocular surface, and support the hypothesis that their activation can be induced by disrupted GC homeostatic responses by microbial stimuli. These findings implicate GC responses in inducing chronic ocular surface damage.
This is a 2021 ARVO Annual Meeting abstract.