Feeling Yu Ting Chen

Interleukin-1 receptor mediates the interplay between CD4+ T cells and ocular resident cells to promote keratinizing squamous metaplasia in Sjögren’s syndrome

Keratinizing squamous metaplasia (SQM) of the ocular mucosal epithelium is a blinding corneal disease characterized by the loss of conjunctival goblet cells (GCs), pathological ocular surface keratinization and tissue recruitment of immune cells. Using the autoimmune regulator (Aire)-deficient mouse as a model for Sjögrens syndrome (SS)-associated SQM, we identified CD4+ T lymphocytes as the main immune effectors driving SQM and uncovered a pathogenic role for interleukin-1 (IL-1). IL-1, a pleiotropic cytokine family enriched in ocular epithelia, governs tissue homeostasis and mucosal immunity. Here, we used adoptive transfer of autoreactive CD4+ T cells to dissect the mechanism whereby IL-1 promotes SQM. CD4+ T cells adoptively transferred from both Aire knockout (KO) and Aire/IL-1 receptor type 1 (IL-1R1) double KO donors conferred SQM to severe-combined immunodeficiency (scid) recipients with functional IL-1R1, but not scid recipients lacking IL-1R1. In the lacrimal gland, IL-1R1 was primarily immunolocalized to ductal epithelium surrounded by CD4+ T cells. In the eye, IL-1R1 was expressed on local mucosal epithelial and stromal cells, but not on resident antigen-presenting cells or infiltrating immune cells. In both tissues, autoreactive CD4+ T-cell infiltration was only observed in the presence of IL-1R1-postive resident cells. Moreover, persistent activation of IL-1R1 signaling led to chronic immune-mediated inflammation by retaining CD4+ T cells in the local microenvironment. Following IL-1R1-dependent infiltration of CD4+ T cells, we observed SQM hallmarks in local tissues—corneal keratinization, conjunctival GC mucin acidification and epithelial cell hyperplasia throughout the ocular surface mucosa. Proinflammatory IL-1 expression in ocular epithelial cells significantly correlated with reduced tear secretion, while CD4+ T-cell infiltration of the lacrimal gland predicted the development of ocular SQM. Collectively, data in this study indicated a central role for IL-1 in orchestrating a functional interplay between immune cells and resident cells of SS-targeted tissues in the pathogenesis of SQM.

Topical Administration of Lacritin Is a Novel Therapy for Aqueous-Deficient Dry Eye Disease

PURPOSE Lacritin is a tear glycoprotein with prosecretory, prosurvival, and mitogenic properties. We examined lacritin levels in the tears of Sjögrens syndrome (SS) patients and explored the therapeutic potential of topical lacritin for the treatment of keratoconjunctivitis sicca. METHODS Tears from healthy controls (n = 14) and SS patients (n = 15) were assayed for lacritin using a C-terminal antibody. In a paired-eye study, autoimmune regulator (Aire) knockout (KO) mice (n = 7) were treated three times daily for 21 days with 10 μL of 4 μM lacritin (left eye) or vehicle (PBS) control (right eye). Tear secretion and ocular surface integrity were assessed at baseline and after treatment. Immunohistochemical staining of CD4+ T cells, cytokeratin-10 (K10), and cytokeratin-12 (K12) expression in the cornea and CD4+ T cell infiltration in the lacrimal glands were assessed. RESULTS Lacritin monomer (421.8 ± 65.3 ng [SS] vs. 655.8 ± 118.9 ng [controls]; P = 0.05) and C-terminal fragment protein (125 ± 34.1 ng [SS] vs. 399.5 ± 84.3 ng [controls]; P = 0.008) per 100 μL of tear eluate were significantly lower in SS patients. In Aire KO mice treated with lacritin, tear secretion increased by 46% (13.0 ± 3.5 mm vs. 8.9 ± 2.9 mm; P = 0.01) and lissamine green staining score significantly decreased relative to baseline (-0.417 ± 0.06 vs. 0.125 ± 0.07; P = 0.02). Expression of K10 but not K12 in the cornea was significantly decreased in lacritin-treated eyes. Focal CD4+ T cell infiltration of the lacrimal glands was significantly reduced on the lacritin-treated side versus the untreated side. CONCLUSIONS Lacritin is significantly reduced in the tears of SS patients. Topically administered lacritin has therapeutic potential for the treatment of aqueous-deficient dry eye disease.

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.

Reduced Levels of Tear Lacritin Are Associated With Corneal Neuropathy in Patients With the Ocular Component of Sjögren's Syndrome.

Purpose To determine whether levels of endogenous tear protein, lacritin, are linked to altered corneal innervation and dry eye severity in patients with Sjögrens syndrome (SS). Methods Clinical data were obtained from 10 SS and 10 age-matched controls. Enzyme-linked immunosorbent assay was used to assess total tear lacritin extracted from Schirmer strips. Western blot was used to detect active lacritin monomer (∼25 kDa), active lacritin fragment (∼12–15 kDa), and inactive tissue transglutaminase–generated lacritin (≥40 kDa). In vivo confocal microscopy was used to assess nerve fiber density (NFD) and length (NFL). Relationships between nerve morphology and tear lacritin were examined by Spearman correlation. Diagnostic performance of tear lacritin was analyzed using receiver operating characteristic. Results Active tear lacritin was significantly reduced in SS patients (3.72 ± 5.62 [SS] versus 18.17 ± 4.57 ng/100 ng total tear protein [controls]; P < 0.001), while inactive lacritin was increased (84.99% ± 11.15% [SS] versus 51.04% ± 12.03% [controls]; P < 0.001). Nerve fiber density (21.70 ± 18.93 vs. 31.80 ± 9.35; P = 0.03) and NFL (4.18 ± 3.44 vs. 6.54 ± 2.47; P < 0.05) were significantly decreased in SS patients compared to controls. Reduced NFL (r = 0.74, P < 0.01) and NFD (r = 0.70, P < 0.01) were highly correlated with reduced tear lacritin. Similarly, total tear lacritin was highly correlated with Schirmers (r = 0.77, P < 0.01), ocular staining (r = −0.80, P < 0.01), and corneal sensitivity (r = 0.81, P < 0.01). Tear lacritin showed equivalent or better diagnostic performance compared to traditional clinical measures for SS (100.00% sensitivity, 85.71% specificity, cutoff = 14.50 ng/100 ng tear protein). Conclusions Reduced tear lacritin levels in SS patients are highly correlated with clinical signs of dry eye, as well as decreased NFD and NFL. Lacritin and its components provide excellent diagnostic sensitivity and specificity in SS.

Aire-deficient mice provide a model of corneal and lacrimal gland neuropathy in Sjögren’s syndrome

Sjögren’s syndrome (SS) is a chronic, autoimmune exocrinopathy that leads to severe dryness of the mouth and eyes. Exocrine function is highly regulated by neuronal mechanisms but little is known about the link between chronic inflammation, innervation and altered exocrine function in the diseased eyes and exocrine glands of SS patients. To gain a better understanding of neuronal regulation in the immunopathogenesis of autoimmune exocrinopathy, we profiled a mouse model of spontaneous, autoimmune exocrinopathy that possess key characteristics of peripheral neuropathy experienced by SS patients. Mice deficient in the autoimmune regulator (Aire) gene developed spontaneous, CD4+ T cell-mediated exocrinopathy and aqueous-deficient dry eye that were associated with loss of nerves innervating the cornea and lacrimal gland. Changes in innervation and tear secretion were accompanied by increased proliferation of corneal epithelial basal cells, limbal expansion of KRT19-positive progenitor cells, increased vascularization of the peripheral cornea and reduced nerve function in the lacrimal gland. In addition, we found extensive loss of MIST1+ secretory acinar cells in the Aire -/- lacrimal gland suggesting that acinar cells are a primary target of the disease, Finally, topical application of ophthalmic steroid effectively restored corneal innervation in Aire -/- mice thereby functionally linking nerve loss with local inflammation in the aqueous-deficient dry eye. These data provide important insight regarding the relationship between chronic inflammation and neuropathic changes in autoimmune-mediated dry eye. Peripheral neuropathies characteristic of SS appear to be tightly linked with the underlying immunopathological mechanism and Aire -/- mice provide an excellent tool to explore the interplay between SS-associated immunopathology and peripheral neuropathy.

miR-205 is a critical regulator of lacrimal gland development

The tear film protects the terrestrial animals 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.