Figure 1.
Keratoconjunctivitis sicca (KCS) and squamous metaplasia (SQM) of the ocular surface in response to CD4+ T cell-mediated autoimmunity.
Representative images of autoimmune-mediated ocular surface disease in a (A) human patient and (B) Aire KO mouse. Aqueous tear deficiency leads to KCS with loss of epithelial integrity indicated by punctate fluorescein staining (A&B-middle panels). SQM is accompanied by pathological keratinization, corneal opacification and vascularization (A&B - right panels). (C) H&E staining of cryosectioned eyes from Aire KO mice reveals representative histological changes associated with disease progression. Open arrows indicate infiltrating immune cells in the corneal stroma.
Figure 2.
Altered lineage commitment of the ocular mucosal epithelium in autoimmune-mediated KCS/SQM.
(A) Immunofluorescent staining of nuclear Pax6 (red, top), corneal-specific CK12 (red, middle) and epidermal-specific CK10 (red, bottom) in the corneal epithelium of WT and Aire KO mice. Scale bar = 50μm (B) TaqMan qPCR analysis of Pax6 gene using corneolimbal epithelial cells from WT and Aire KO mice and impression cytology specimens from healthy human control (Con) and chronic KCS (KCS) patients. Pax6 expression in WT mice or healthy control patients was used as the reference (designated 1-fold) to generate relative quantitation (RQ) values. Data are shown as mean RQ±SE. Seven mice were studied per group, as well 15 KCS patients and 7 healthy controls. Unpaired T-test (mouse) or Wilcoxan Rank Sum test (human) was used to test for differences between groups, with P<0.05 (*) and P<0.01 (**) considered statistically significant. (C) Cytospin was used to assess single-cell expression of Pax6 (red, upper panels), CK12 (brown, middle panels) and epidermal cytokeratin K10 (red, bottom panels) in WT and KO mice. Arrows and arrowheads indicate positive and negative stained cells, respectively. (D) Quantitative analysis of cytospin specimens expressed as the percentage of cells staining positive for Pax6, CK12 and CK10. Data were generated by analyzing three separate fields of view from an individual specimen with a minimum of three animals analyzed per group. Group mean comparisons were assessed by T-test, with P<0.05 (*) and P<0.01 (**) considered statistically significant.
Figure 3.
Role of IL-1R1/IL-1 signaling in mediating Pax6 loss and altered lineage commitment in autoimmune-mediated KCS/SQM.
(A) Adoptive transfer (AT) experimental design. (B) Transcriptional profile of corneolimbal epithelial Pax6 in the four AT groups with control group (WT scid) set as the reference (designated 1-fold) to generate relative quantitation (RQ) values of Pax6 expression. Data are shown as mean RQ value ± SE; statistically significant differences are noted as P<0.01 (**); n=5 per group. (C) Immunolocalization studies of Pax6 staining in the four AT groups (red); Scale bar = 50μm. (D) IL-1β and Pax6 transcript levels in WT and Aire KO mice expressed as mean ± SE RQ values where an arbitrary WT control mouse was set as the reference (designated 1-fold); P<0.05 (*) indicates statistical significance; n = 3 per group. Regression analysis of transcript levels indicates Pax6 and IL-1β expression are negatively correlated in Aire KO mice.
Figure 4.
Cytokeratin expression profile of the corneolimbal epithelium in autoimmune-mediated KCS/SQM.
(A) Transcriptional profiling of ocular surface cytokeratin expression in corneolimbal epithelial cells isolated from WT and Aire KO mice. Quantitative PCR for CK12, CK10, SPRR1B and CK14 were expressed as relative quantitation (RQ) values where an arbitrary WT control mouse was set as the reference (designated 1-fold). RQ values are shown as mean ± SE; n=3 mice per group. Unpaired T-test (CK12 and CK14) or the Kruskal-Wallis Rank Sum test (SPRR1B and CK10) was used to test for differences between WT vs. Aire KO with P<0.05 (*) and P<0.01 (**) considered statistically significant. (B) Downregulation of Pax6 predicts the cytokeratin maturation profile of corneolimbal epithelial cells. Regression analysis shows Pax6 was positively correlated with CK12 but negatively correlated with CK10, CK14 and SPRR1B. R2 values are provided.
Figure 5.
Pax6 adenovirus restores corneal phenotype in autoimmune-mediated KCS/SQM.
(A) Immunostaining of corneal Pax6 (red), CK12 (green), and CK10 (green) in Aire KO mice 5 days after injection with Pax6-adenovirus (Pax6-Adeno) or control adenovirus (Con-Adeno). Images are representative of three (Con-Adeno) and five (Pax6-Adeno) mice per group. Nuclei is stained blue with DAPI. (B) Linear regression indicates cytokeratin switch from CK12 (top) to CK10 (bottom) was highly correlated to recovery of Pax6 following forced expression.