10 research outputs found
Pax6 downregulation mediates abnormal lineage commitment of the ocular surface epithelium in aqueous-deficient dry eye disease.
Keratinizing squamous metaplasia (SQM) of the ocular surface is a blinding consequence of systemic autoimmune disease and there is no cure. Ocular SQM is traditionally viewed as an adaptive tissue response during chronic keratoconjunctivitis sicca (KCS) that provokes pathological keratinization of the corneal epithelium and fibrosis of the corneal stroma. Recently, we established the autoimmune regulator-knockout (Aire KO) mouse as a model of autoimmune KCS and identified an essential role for autoreactive CD4+ T cells in SQM pathogenesis. In subsequent studies, we noted the down-regulation of paired box gene 6 (Pax6) in both human patients with chronic KCS associated with Sjögren's syndrome and Aire KO mice. Pax6 encodes a pleiotropic transcription factor guiding eye morphogenesis during development. While the postnatal function of Pax6 is largely unknown, we hypothesized that its role in maintaining ocular surface homeostasis was disrupted in the inflamed eye and that loss of Pax6 played a functional role in the initiation and progression of SQM. Adoptive transfer of autoreactive T cells from Aire KO mice to immunodeficient recipients confirmed CD4+ T cells as the principal downstream effectors promoting Pax6 downregulation in Aire KO mice. CD4+ T cells required local signaling via Interleukin-1 receptor (IL-1R1) to provoke Pax6 loss, which prompted a switch from corneal-specific cytokeratin, CK12, to epidermal-specific CK10. The functional role of Pax6 loss in SQM pathogenesis was indicated by the reversal of SQM and restoration of ocular surface homeostasis following forced expression of Pax6 in corneal epithelial cells using adenovirus. Thus, tissue-restricted restoration of Pax6 prevented aberrant epidermal-lineage commitment suggesting adjuvant Pax6 gene therapy may represent a novel therapeutic approach to prevent SQM in patients with chronic inflammatory diseases of the ocular surface
Pax6 Downregulation Mediates Abnormal Lineage Commitment of the Ocular Surface Epithelium in Aqueous-Deficient Dry Eye Disease
Keratinizing squamous metaplasia (SQM) of the ocular surface is a blinding consequence of systemic autoimmune disease and there is no cure. Ocular SQM is traditionally viewed as an adaptive tissue response during chronic keratoconjunctivitis sicca (KCS) that provokes pathological keratinization of the corneal epithelium and fibrosis of the corneal stroma. Recently, we established the autoimmune regulator-knockout (Aire KO) mouse as a model of autoimmune KCS and identified an essential role for autoreactive CD4+ T cells in SQM pathogenesis. In subsequent studies, we noted the down-regulation of paired box gene 6 (Pax6) in both human patients with chronic KCS associated with Sjögren’s syndrome and Aire KO mice. Pax6 encodes a pleiotropic transcription factor guiding eye morphogenesis during development. While the postnatal function of Pax6 is largely unknown, we hypothesized that its role in maintaining ocular surface homeostasis was disrupted in the inflamed eye and that loss of Pax6 played a functional role in the initiation and progression of SQM. Adoptive transfer of autoreactive T cells from Aire KO mice to immunodeficient recipients confirmed CD4+ T cells as the principal downstream effectors promoting Pax6 downregulation in Aire KO mice. CD4+ T cells required local signaling via Interleukin-1 receptor (IL-1R1) to provoke Pax6 loss, which prompted a switch from corneal-specific cytokeratin, CK12, to epidermal-specific CK10. The functional role of Pax6 loss in SQM pathogenesis was indicated by the reversal of SQM and restoration of ocular surface homeostasis following forced expression of Pax6 in corneal epithelial cells using adenovirus. Thus, tissue-restricted restoration of Pax6 prevented aberrant epidermal-lineage commitment suggesting adjuvant Pax6 gene therapy may represent a novel therapeutic approach to prevent SQM in patients with chronic inflammatory diseases of the ocular surface
miR-205 is a critical regulator of lacrimal gland development.
The tear film protects the terrestrial animal's ocular surface and the lacrimal gland provides important aqueous secretions necessary for its maintenance. Despite the importance of the lacrimal gland in ocular health, molecular aspects of its development remain poorly understood. We have identified a noncoding RNA (miR-205) as an important gene for lacrimal gland development. Mice lacking miR-205 fail to properly develop lacrimal glands, establishing this noncoding RNA as a key regulator of lacrimal gland development. Specifically, more than half of knockout lacrimal glands never initiated, suggesting a critical role of miR-205 at the earliest stages of lacrimal gland development. RNA-seq analysis uncovered several up-regulated miR-205 targets that may interfere with signaling to impair lacrimal gland initiation. Supporting this data, combinatorial epistatic deletion of Fgf10, the driver of lacrimal gland initiation, and miR-205 in mice exacerbates the lacrimal gland phenotype. We develop a molecular rheostat model where miR-205 modulates signaling pathways related to Fgf10 in order to regulate glandular development. These data show that a single microRNA is a key regulator for early lacrimal gland development in mice and highlights the important role of microRNAs during organogenesis
Cytokeratin expression profile of the corneolimbal epithelium in autoimmune-mediated KCS/SQM.
<p>(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>B</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. R<sup>2</sup> values are provided.</p
Keratoconjunctivitis sicca (KCS) and squamous metaplasia (SQM) of the ocular surface in response to CD4+ T cell-mediated autoimmunity.
<p>Representative images of autoimmune-mediated ocular surface disease in a (<b>A</b>) human patient and (<b>B</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). (<b>C</b>) 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. </p
Pax6 adenovirus restores corneal phenotype in autoimmune-mediated KCS/SQM.
<p>(<b>A</b>) 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>B</b>) Linear regression indicates cytokeratin switch from CK12 (top) to CK10 (bottom) was highly correlated to recovery of Pax6 following forced expression. </p
Role of IL-1R1/IL-1 signaling in mediating Pax6 loss and altered lineage commitment in autoimmune-mediated KCS/SQM.
<p>(<b>A</b>) Adoptive transfer (AT) experimental design. (<b>B</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. (<b>C</b>) Immunolocalization studies of Pax6 staining in the four AT groups (red); Scale bar = 50μm. (<b>D</b>) 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. </p