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Ying-Ting Chen, Nancy McNamara, cornea; Downregulation of master transcription factor, Pax6, contributes to autoimmune-mediated keratinizing squamous metaplasia of the ocular surface via the aberrant activation of basal progenitor cells. Invest. Ophthalmol. Vis. Sci. 2013;54(15):983.
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© ARVO (1962-2015); The Authors (2016-present)
To study the role of ocular master regulator, Pax6, in the dysregulation of keratinocyte progenitor cells (PCs) during T cell-mediated keratoconjunctivitis sicca (KCS) with keratinizing squamous metaplasia (SQM).
Aire KO mice and immunodeficient (scid) recipients of Aire KO-derived autoreactive CD4+T cells were used as models of autoimmune SQM. Proliferative activity was measured by the density of Ki67+ basal cells, designated as the mitotic index (MI). The relative length of S phase in the 4-phase cell cycle was obtained by dividing the density of BrdU+ cells (labeled by a single BrdU pulsing) by the MI. Activated PCs, transient amplifying cells (TACs), postmitotic cells (PMCs), young terminally differentiated epithelial cells (TDCs) and immune effector cells were labeled by continuous BrdU pulsing for 7 days. Label-retaining cells including slow-cycling SCs, long-lived PMC/TDCs and memory T cells were visualized after a 21-day BrdU. Local adenoviral transfer of Pax6 was employed to assess Pax6’s modulatory effects on PC’s self-renewal and differentiation in Aire KO’s SQM tissue.
Compared to wild type mice, the MI of p63+ PCs increased 3.1±1.2 fold in the limbus and cornea of Aire KO mice. Such an increase in proliferation was accompanied by a 42% decrease in Pax6 transcripts, a 52% decrease in scid recipients of Aire KO-derived CD4+ T, and an 83% decrease in clonal cells of Aire KO PCs cultivated on 3T3 feeders (p < 0.05, n=5 per group). BrdU incorporation kinetics revealed a 1.9-fold, 2.1-fold, and 0.5-fold labeling index in Aire KO mice following 1-shot, 7-day pulse and 21-day chase protocols, respectively (all p < 0.05 vs WT at 1-fold). Together, these results suggested downregulation of Pax6 is associated with an accelerated epithelial turnover and a relatively shorter S phase in rapid cycling cells. Adenoviral induction of Pax6 in Aire KO basal PCs restored the corneal phenotype.
The downregulation of Pax6 in basal PCs during ocular autoimmune SQM is mediated by PC- and limbal niche-reactive CD4+ T effectors. Restoration of Pax6 reverses aberrant epidermal-lineage commitment suggests adjuvant Pax6 gene therapy may compliment immunosuppressants as a novel therapeutic approach to manage SQM disease in sicca patients
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