Abstract
Sjögren syndrome (SS) is an autoimmune condition that targets the salivary and lacrimal glands, with cardinal clinical signs of dry eye (keratoconjunctivitis sicca, KCS) and dry mouth. The conjunctiva of SS patients is often infiltrated by immune cells that participate in the induction and maintenance of local inflammation. The purpose of this study was to investigate immune-related molecular pathways activated in the conjunctiva of SS patients. Female SS patients (n=7) and controls (n=19) completed a series of oral, ocular surface exams. Symptom severity scores were evaluated using validated questionnaires (OSDI and SANDE). All patients fulfilled the ACR/EULAR criteria for SS and the criteria for KCS. Fluorescein and lissamine green dye staining evaluated tear-break-up time (TBUT), corneal and conjunctival disease, respectively. Impression cytology of the temporal bulbar conjunctiva was performed to collect cells lysed and subjected to gene expression analysis using the NanoString Immunology Panel. 53/594 differentially expressed genes (DEGs) were observed between SS and healthy controls; 49 DEGs were upregulated, and 4 were downregulated (TRAF5, TGFBI, KLRAP1, and CMKLRI). The top 10 DEGs in descending order were BST2, IFITM1, LAMP3, CXCL1, IL19, CFB, LY96, MX1, IL4R, CDKN1A. Twenty pathways had a global significance score greater or equal to 2. Spearman correlations showed that 29/49 upregulated DEGs correlated with either TBUT (inverse) or OSDI or conjunctival staining score (positive correlations). Venn diagrams identified that 26/29 DEGs correlated with TBUT, 5/26 DEGs correlated with OSDI, and 16/26 correlated with conjunctival staining scores. Five upregulated DEGs (CFB, CFI, IL1R1, IL2RG, IL4R) were uniquely negatively correlated with TBUT. These data indicate that the conjunctiva of SS patients exhibits a phenotype of immune activation, although some genes could be inhibitory. Some of the DEGs and pathways overlap with previous DEGs in salivary gland biopsies, but new DEGs were identified, and some of these correlated with symptoms and signs of dry eye. Our results indicate that gene analysis of conjunctiva imprints is a powerful tool to understand the pathogenesis of SS and develop new therapeutic targets.
Highlights
Patients with Sjögren syndrome (SS) develop an ocular surface disease called keratoconjunctivitis sicca (KCS)
We and others have shown that impression cytology of the conjunctiva collects a mix of epithelial and immune cells [8, 14, 33]
To gain insights into the molecular mechanisms involved locally, impression cytology of the conjunctiva was used to collect conjunctival cells, which were lysed and processed for RNA analysis using a NanoString® nCounter panel enriched for immune genes (Immunology V2)
Summary
Patients with Sjögren syndrome (SS) develop an ocular surface disease called keratoconjunctivitis sicca (KCS). Pathological changes in the conjunctiva in KCS include altered epithelial differentiation with increased expression of cornified envelope precursors [1] that are found in the epidermis (involucrin, SPRR2G) [2, 3] and dysfunction and loss of the goblet cells that secrete gel-forming mucin that is essential for maintaining tear film stability [4]. Current methods to diagnose SS KCS include the use of dyes that stain conjunctival epithelial cells lacking a mucin coating or impression cytology to measure conjunctival goblet cell density, but they do not measure disease-relevant biomarkers. Interferon IFN-g disrupts cholinergic mediated secretion and causes unfolded protein response (UPR) and apoptosis of conjunctival goblet cells [11, 12]. Additional inflammatory mediators have been detected in the conjunctiva [1, 13, 14]
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