Molecular and Functional Characterization of Ocular Progenitors in Autoimmune-mediated Keratinizing Squamous Metaplasia of the Ocular Surface Mucosa

Abstract

 

Purpose:

 

Ocular keratinizing squamous metaplasia (SQM) is a blinding consequence of many autoimmune diseases that affect the ocular surface. It is traditionally viewed as an adaptive tissue response to chronic ocular dryness that provokes pathological keratinization and opacification of the cornea. Using the autoimmune regulator (Aire) knockout (KO) mouse, we sought to characterize the topographical, phenotypical, and cell cycle alterations in ocular progenitors in chronic CD4+ T cell-mediated autoimmune SQM.

 

Methods:

 

H&E staining and PAS staining were used to evaluate the corneal histology and goblet cell changes in SQM progression. Lectin staining to MAA and SNA was used to evaluate sialyl changes of ocular surface mucoglycan. Immunostaining to CD4 and p63 was used to localize autoreactive T cells and ocular progenitors. RT-qPCR was employed to quantify the expression of corneal lineage K12 versus epidermal lineage marker K10. SPRR1b immunostaining was used to assess keratinization. BrdU injection to Aire-KO mice for 7 days was used to study cell cycle in stem cell pool. 3T3-based clonal culture was used to characterize SQM-derived stem cells in vitro.

 

Results:

 

Our studies revealed (i) hyperplasia of the ocular mucosal epithelium (105+/- 25 in Aire-KO vs. 46+/-14 μm in WT mice); (ii) a shift from corneal (K12-expressing) to epidermal (K10-expressing) phenotype; (iii) a decrease in PAS+ goblet cells; (iv) Increased sialylation of ocular mucoglycans in peripheral corneal, limbus and conjunctiva; and (v) pathological keratinization of the ocular mucosal surface. Here, we provide evidence to suggest the etiology of autoimmune SQM results from pathological alteration of epithelial progenitor cells (ePCs). Focal aggregation of infiltrating autoreactive CD4+ T cells target two distinct ocular progenitor niches: the limbus and the GC-rich zone of the conjunctiva. BrdU labeling in Aire KO mice revealed a hyperproliferative growth kinetic in p63+ ePCs that are in lack of Pax6 expression. Quantitative PCR for Pax6 showed a 51 +/- 15% decrease in Aire-KO vs WT mice (p<0.01). p63+ basal cells revealed a patchy pattern in Aire-KO in contrast to a continuous expression pattern in WT. 3T3-based clonal analysis of ePCs revealed a merocloanl dormancy in WT versus the paraclonal dormancy with spontaneous limbal spheroid formation in Aire-KO.

 

Conclusions:

 

Our data suggest a role for dysregulated Pax6 transcriptional activity in p63 ePCs for cell cycle progression and lineage commitment of autoimmune SQM.

 

Keywords: autoimmune disease • cornea: epithelium • pathobiology